Synthesis of clasto-lactacystin β-lactone and analogs thereof

ABSTRACT

The present invention is directed to an improved synthesis of clasto-lactacystin-β-lactone, and analogs thereof, that proceeds in fewer steps and in much greater overall yield than syntheses described in the prior art. The synthetic pathway relies upon a novel stereospecific synthesis of an oxazoline intermediate and a unique stereoselective addition of a formyl amide to the oxazoline. Also described are novel clasto-lactacystin-β-lactones, and analogs thereof and their use as proteosome inhibitors.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.09/597,514, filed on Jun. 20, 2000 now U.S. Pat. No. 6,294,560, which isa divisional of U.S. patent application Ser. No. 09/134,674, filed onAug. 14, 1998, now U.S. Pat. No. 6,133,308, which in turn claimspriority form U.S. Provisional Patent Application Serial No. 60/055,848,filed on Aug. 15, 1997, and U.S. Provisional Patent Application SerialNo. 60/067,352, filed on Dec. 3, 1997, all of which applications arehereby incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates generally to methods for preparing lactacystin andrelated compounds, to novel analogs of lactacystin andclasto-lactacystin β-lactone, and their uses as proteasome inhibitors.

2. Description of Related Art

The Streptomyces metabolite lactacystin (1) inhibits cell cycleprogression and induces neurite outgrowth in cultured neuroblastomacells (Omura et al., J. Antibiotics 44:117 (1991); Omura et al., J.Antibiotics 44:113 (1991); Fenteany et al., Proc. Natl. Acad. Sci. (USA)91:3358 (1994)). The cellular target mediating these effects is the 20Sproteasome, the proteolytic core of the 26S proteasome, which is thecentral component of the ubiquitin-proteasome pathway for intracellularprotein degradation. Mechanistic studies have established thatlactacystin inhibits the proteasome through the intermediacy of theactive species, clasto-lactacystin β-lactone (2), which specificallyacylates the N-terminal threonine residue of the proteasome X/MB1subunit (Fenteany, et al., Science 268:726 (1995); Dick et al., J. Biol.Chem. 271:7273 (1996)). Lactacystin analogs are disclosed by Fenteany etal. (WO 96/32105).

The ubiquitin-proteasome pathway is involved in a variety of importantphysiological processes (Goldberg et al., Chemistry & Biology 2:503(1995); Ciechanover Cell 79:13 (1994); Deshaies, Trends Cell Biol.5:43 1(1995)). In fact, the bulk of cellular proteins are hydrolyzed by thispathway. Protein substrates are first marked for degradation by covalentconjugation to multiple molecules of a small protein, ubiquitin. Theresultant polyubiquitinated protein is then recognized and degraded bythe 26S proteasome. Long recognized for its role in degradation ofdamaged or mutated intracellular proteins, this pathway is now alsoknown to be responsible for selective degradation of various regulatoryproteins. For example, orderly cell cycle progression requires theprogrammed ubiquitination and degradation of cyclins. Theubiquitin-proteasome pathway also mediates degradation of a number ofother cell cycle regulatory proteins and tumor suppressor proteins(e.g., p21, p27, p53). Activation of the transcription factor NF-κB,which plays a central role in the regulation of genes involved in theimmune and inflammatory responses, is dependent upon ubiquitination anddegradation of an inhibitory protein, IκB-α (Palombella et al., WO95/25533). In addition, the continual turnover of cellular proteins bythe ubiquitin-proteasome pathway is essential to the processing ofantigenic peptides for presentation on MHC class I molecules (Goldbergand Rock, WO 94/17816).

The interesting biological activities of lactacystin andclasto-lactacystin β-lactone and the scarcity of the natural materials,as well as the challenging chemical structures of the molecules, havestimulated synthetic efforts directed toward lactacystin and relatedanalogs. Corey and Reichard J. Am. Chem. Soc. 114:10677 (1992);Tetrahedron Lett. 34:6977 (1993)) achieved the first total synthesis oflactacystin, which proceeded in 15 steps and 10% overall yield. The keyfeature of the synthesis is a stereoselective aldol reaction of acis-oxazolidine aldehyde derived from N-benzylserine to construct theC(6)-C(7) bond. In the synthesis reported by (Uno et al., J. Am. Chem.Soc. 116:2139 (1994)), stereo selective Mukaiyama-aldol reaction of abicyclic oxazolidine silyl enol ether intermediate derived fromD-pyroglutamic acid is employed in C(5)-C(9) bond construction. Thissynthesis proceeds in 19 steps and 5% overall yield. Aldol reactionsunder basic conditions of a similar bicyclic oxazolidine intermediateform the basis of model studies reported by (Dikshit et al., TetrahedronLett. 36:6131 (1995)).

Aldol reactions of oxazoline-derived enolates feature prominently in thesynthesis of lactacystin reported by Smith and coworkers (Suazuka etal., J. Am. Chem. Soc. 115:5302 (1993); Nagamitsu et al., J. Am. Chem.Soc. 118:3584 (1996)) and in the synthesis of (6R)-lactacystin reportedby (Corey and Choi Tetrahedron Lett. 34:6969 (1993)); Choi Ph.D.,Thesis, Harvard University, 44 (1995). In the former synthesis, whichproceeds in 20 steps and 9% overall yield, the enolate is condensed withformaldehyde to install a single carbon atom, which must then beelaborated in a number of additional steps. In the Corey and Choisynthesis, the aldol reaction selectively provides the product ofundesired stereochemistry, resulting in the eventual preparation of theC(6) epimer of lactacystin, which is devoid of biological activity.

Lactacystin has also been prepared in 22 steps and 2% overall yield fromD-glucose (Chida et al., J. Chem. Soc., Chem. Commun. 793 (1995)). Thebiosynthetic pathway involved in production of the natural product hasbeen investigated in feeding experiments involving ¹³C-enrichedcompounds (Nakagawa et al., Tetrahedron Lett. 35:5009 (1994)).

The reported syntheses of lactacystin are lengthy and proceed in lowyield. Furthermore, none of these syntheses is readily adapted foranalog synthesis. Thus, there is a need for improved methods forpreparing lactacystin, clasto-lactacystin β-lactone, and analogs thereof

SUMMARY OF THE INVENTION

A first aspect of the present invention relates to a process for forminglactacystin or analogs thereof having Formula VI or clasto-lactacystinβ-lactone or analogs thereof having Formula VII:

wherein

R¹ is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkaryl, aralkyl, wherethe ring portion of any of said aryl, aralkyl, or alkaryl can beoptionally substituted;

R² is alkyl, cycloalkyl, aryl, alkaryl, aralkyl, alkoxy, hydroxy,alkoxyalkyl, or amido, where the ring portion of any of said aryl,aralkyl, or alkaryl can be optionally substituted; and

R⁷ is alkyl, aryl, aralkyl, alkaryl, wherein any of said alkyl, aryl,aralkyl or alkaryl can be optionally substituted.

A second aspect of the present invention is directed to a method offorming formyl amides of Formula XIV:

where R² is alkyl, cycloalkyl, aryl, alkaryl, aralkyl, alkoxy, hydroxy,alkoxyalkyl, or amido, where the ring portion of any of said aryl,aralkyl, or alkaryl can be optionally substituted; and

R⁵ and R⁶ are independently one of alkyl or alkaryl; or R⁵ and R⁶ whentaken together with the nitrogen atom to which they are attached form a5- to 7-membered heterocyclic ring, which may be optionally substituted,and which optionally may include an additional oxygen or nitrogen atom.

A third aspect of the present invention relates to formingtri-substituted oxazolines of Formula Ia or Ib:

where R¹ is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkaryl, aralkyl,where the ring portion of any of said aryl, aralkyl, or alkaryl can beoptionally substituted; and R⁴ is aryl or heteroaryl, either of whichmay be optionally substituted. The tri-substituted oxazolines ofFormulae Ia and Ib are useful as starting materials in forminglactacysin, clasto-lactacystin β-lactone or analogs thereof via theprocess described herein.

A fourth aspect of the present invention is directed to lactacysin,clasto-lactacystin β-lactone or analogs of Formulae VI and VI thatpossess unexpected biological activity. Lactacystin, clasto-lactacystinβ-lactone, and analogs thereof possess biological activity as inhibitorsof the proteasome. They can be used to treat conditions mediateddirectly by the function of the proteasome, such as muscle wasting, ormediated indirectly via proteins which are processed by the proteasome,such as the transcription factor NF-κB.

A fifth aspect of the present invention relates to pharmaceuticalcompositions, comprising a compound of Formula VI or Formula VII, and apharmaceutically acceptable carrier or diluent.

A sixth aspect of the present invention relates to methods of inhibitingproteasome function or treating a condition that is mediated directly orindirectly by the function of the proteasome, by administering acompound of Formula VI or Formula VII that possesses unexpectedly highactivity in inhibiting the proteasome. Preferred Embodiments aredirected to the use of a compound of Formulae VI or VII to prevent orreduce the size of infarct after vascular occlusion for example, fortreating neuronal loss following stroke. An additional preferredembodiment is directed to the use of said compounds for treating asthma.

A seventh aspect of the invention relates to enantiomerically-enrichedcompositions of formyl amides of Formula XIV.

An eighth aspect of the present invention relates to novel individualintermediates, such as aldols of Formula II and aminodiols of FormulaIII:

and individual steps within the multistep process for forminglactacystin, clasto-lactacystin β-lactone or various analogs thereof.

A ninth aspect of the present invention relates to individualintermediates, such as compounds of Formulae XVII, XVIII and XIX:

where X is a halogen, preferably Cl, Br or I, as well as individualsteps within the multistep process for forming substituted oxazolines ofFormula I.

Other features or advantages of the present invention will be apparentfrom the following detailed description, and also from the appendingclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. depicts a graph showing the effect of compound 3b, administeredi.v., on infarct volume in rats (n=6-8).

FIG. 2. depicts a graph showing the effect of compound 3b, administeredi.v. on neurological score in rats (n=6-8).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to an improved multi-step synthesis oflactacystin, clasto-lactacystin β-lactone, and analogs thereof, thatproceeds in fewer steps and in much greater overall yield than synthesesdescribed in the prior art. A number of individual process steps andchemical intermediates distinguish this synthetic pathway from pathwaysdescribed in the prior art. For example, this synthetic pathway reliesupon a novel stereospecific synthesis of an oxazoline intermediate, anda unique stereoselective addition of a formyl amide to the oxazoline.

The invention is also directed to novel analogs of Formulae VI and VIIthat possess unexpected biological activity. Lactacystin,clasto-lactacystin β-lactone, and analogs thereof possess biologicalactivity as inhibitors of the proteasome. They can be used to treatconditions mediated directly by the function of the proteasome, such asmuscle wasting, or mediated indirectly via proteins which are processedby the proteasome, such as the transcription factor NF-κB. The presentinvention is also directed to methods of inhibiting proteasome functionor treating a condition that is mediated directly or indirectly by thefunction of the proteasome, by administering a compound of Formula VI orVII that possesses unexpectedly high activity in inhibiting theproteasome. In a preferred aspect of the invention, a pharmaceuticalcomposition that includes a compound of Formula VI or Formula VII isadministered to treat ischemic or reperfusion injury. For example, in apreferred embodiment said compounds can be used to treat, prevent orameliorate neuronal loss following stroke.

Synthetic Processes

A first aspect of the present invention relates to processes for forminglactacystin and analogs thereof having Formula VI and clasto-lactacystinβ-lactone and analogs thereof having Formula VII:

wherein

R¹ is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkaryl, aralkyl, wherethe ring portion of any of said aryl, aralkyl, or alkaryl can beoptionally substituted;

R² is alkyl, cycloalkyl, aryl, alkaryl, aralkyl, alkoxy, hydroxy,alkoxyalkyl, or amido, where the ring portion of any of said aryl,aralkyl, or alkaryl can be optionally substituted; and

R⁷ is alkyl, aryl, aralkyl, alkaryl, wherein any of said alkyl, aryl,aralkyl or alkaryl can be optionally substituted.

The processes for forming these compounds rely upon formation of acommon carboxylic acid intermediate of Formula V:

where R¹ and R² are as defined above for Formulae VI and VII. Thesesteps include:

(a) deprotonating a substituted aryl or heteroaryl oxazoline of FormulaI:

where R¹ is as defined above, and R³ is alkyl, alkenyl, alkynyl,cycloalkyl, aryl, alkaryl, any of which can be optionally substituted;and

R⁴ is aryl or heteroaryl, either of which may be optionally substituted;by treating said substituted aryl or heteroaryl oxazoline with a strongbase to form an enolate;

(b) transmetallating said enolate with a metal selected from the groupconsisting of titanium, aluminum, tin, zinc, magnesium and boron, andthereafter treating with a formyl amide of Formula XIV:

where R² is as defined above for Formulae VI and VII, and R⁵ and R⁶ areindependently one of alkyl or alkaryl; or R⁵ and R⁶ when taken togetherwith the nitrogen atom to which they are attached form a 5- to7-membered heterocyclic ring, which may be optionally substituted, andwhich optionally may include an additional oxygen or nitrogen atom, toform an adduct of Formula II:

where R¹ through R⁶ are as defined above;

c) catalytically hydrogenating said adduct of Formula II to form aγ-lactam of Formula IV:

where R¹, R² and R³ are as defined above;

d) saponifying said γ-lactam of Formula IV to form a lactam carboxylicacid of Formula V:

where R¹ and R² are as defined above.

The carboxylic acid intermediate of Formula V can be cyclized bytreatment with a cyclizing reagent to form aclasto-lactacystin-β-lactone or analog thereof of Formula VII, which canbe optionally further reacted with a thiol (R⁷SH), such asN-acetylcysteine, to form lactacystin or an analog thereof havingFormula VI.

Alternatively, the carboxylic acid intermediate of Formula V can bedirectly coupled to a thiol (R⁷SH), such as N-acetylcysteine, to formlactacystin or an analog thereof having Formula VI.

A second aspect of the present invention relates to the formation ofenantiomerically-enriched formyl amides of Formula XIV:

wherein R², R⁵ and R⁶ are as defined above, said method comprising:

(a) deprotonating a compound of Formula VIII:

where R⁸ is isopropyl or benzyl, and thereafter acylating the resultantanion with R²CH₂COCl to form an acyloxazolidinone of Formula IX:

where R² and R⁸ are as defined above;

(b) stereoselectively reacting the acyloxazolidinone of Formula IX withbenzyloxymethyl chloride to form a protected alcohol of Formula X:

where R² and R⁸ are as defined above;

(c) hydrolyzing the protected alcohol of Formula X to form a carboxylicacid of Formula XI:

where R² is as defined above;

(d) coupling said acid of Formula XI with an amine R⁵R⁶NH₂ to provide anamide of Formula XII:

where R², R⁵ and R⁶ are as defined above;

(e) catalytically hydrogenating, the amide of Formula XII to form analcohol of Formula XIII:

where R², R⁵ and R⁶ are as defined above; and

(f) oxidizing the resultant alcohol of Formula XIII to give a formylamide of Formula XIV.

A third aspect of the invention relates to a process for forming atri-substituted cis-oxazoline compound of Formula Ia:

wherein

R¹ is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkaryl, aralkyl, wherethe ring portion of any of said aryl, aralkyl, or alkaryl can beoptionally substituted;

R³ is alkyl, cycloalkyl, aryl, alkaryl, any of which can be optionallysubstituted; and

R⁴ is aryl or heteroaryl, either of which may be optionally substituted;said method comprising:

(a) asymmetrically dihydroxylating an alkene intermediate of Formula XV:

to form an optically active diol of Formula XVIa:

(b) reacting said optically active diol of Formula XVIa with anorthoester derived from an aromatic carboxylic acid under acid catalysis(Lewis or Brönsted acid) to give a mixed orthoester, and thereafterreacting the resulting mixed orthoester intermediate with a reagentselected from the group consisting of lower alkanoyl halides, hydrohalicacids (HX, where X is a halogen), acid chlorides, and halogen-containingLewis acids (for example BBr₃, SnCl₄, Ti(OR)₂Cl₂, Ti(OR)₃Cl, Me₃SiX,where X is a halogen, and the like) in the presence of a base to form aderivative of Formula XVIIa:

wherein X is a halogen, preferably Cl, Br or I;

(c) reacting said derivative of Formula XVIIa with an alkali metal azideto form an azide of Formula XVIIIa:

(d) catalytically hydrogenating said azide to form a compound of FormulaXIXa:

(e) subjecting the compound of Formula XIXa to ring closing conditionsto form said substituted aryl- or heteroaryloxazoline of Formula I withinversion of configuration at the oxygen-substituted carbon to produce acis-oxazoline of Formula Ia; wherein for each of Formulae XV, XVIa,XVIIa, XVIIIa and XIXa, R¹, R³ and R⁴ are as defined above for FormulaI.

Alternatively, the third aspect of the invention relates to a processfor forming a tri-substituted trans-oxazoline compound of Formula Ibcomprising:

(a) asymmetrically dihydroxylating an alkene intermediate of Formula XV:

to form an optically active diol of Formula XVIb:

(b) reacting said optically active diol of Formula XVIb with anorthoester derived from an aromatic carboxylic acid under acid catalysis(Lewis or Brönsted acid) to give a mixed orthoester, and thereafterreacting the resulting mixed orthoester intermediate with a reagentselected from the group consisting of lower alkanoyl halides, hydrohalicacids (HX, where X is halogen), acid chlorides, and halogen-containingLewis acids (for examples, BBr₃, SnCl₄, Ti(OR)₂Cl₂, Ti(OR)₃Cl, Me₃SiX,where X is a halogen, and the like) in the presence of a base to form aderivative of Formula XVIIb:

wherein X is a halogen, preferably Cl, Br, or I;

(c) reacting said derivative of Formula XVIIb with an alkali metal azideto form an azide of Formula XVIIIb:

(d) catalytically hydrogenating said azide to form a compound of FormulaXIXb:

(e) subjecting the compound of Formula XIXb to ring closing conditionsto form said substituted aryl- or heteroaryloxazoline of Formula Ib,wherein the ring closure reaction proceeds with retention ofconfiguration at the oxygen-substituted carbon to produce atrans-oxazoline of Formula Ib; wherein for each of Formulae XV, XVI,XVII, XVIII and XIX, R¹, R³, and R⁴ are as defined above for Formula I.

With respect to the processes described above, the following preferredvalues are applicable:

Preferred values of R¹ are C₁₋₁₂alkyl, especially C₁₋₈alkyl,C₃₋₈cycloalkyl, especially C₃₋₆cycloalkyl, C₂₋₈alkenyl, C₂₋₈alkynyl,C₆₋₁₄aryl, especially C₆₋₁₀aryl, C₆₋₁₀ar(C₁₋₆)alkyl orC₁₋₆alk(C₆₋₁₀)aryl, where the ring portion of any of said aryl, aralkyl,or alkaryl can be optionally substituted. Substituents that can beoptionally present on the aryl ring of an R¹ moiety include one or more,preferably one or two, of hydroxy, nitro, trifluoromethyl, halogen,C₁₋₆alkyl, C₆₋₁₀aryl, C₁₋₆alkoxy, C₁₋₆aminoalkyl, C₁₋₆aminoalkoxy,amino, C₂₋₆alkoxycarbonyl, carboxy, C₁₋₆hydroxyalkyl, C₂₋₆hydroxyalkoxy,C₁₋₆alkylsulfonyl, C₆₋₁₀arylsulfonyl, C₁₋₆alkylsulfinyl,C₁₋₆alkylsulfonamido, C₆₋₁₀arylsulfonamido, C₆₋₁₀ar(C₁₋₆)alkylsulfonamido, C₁₋₆alkyl, C₁₋₆hydroxyalkyl, C₆₋₁₀aryl, C₆₋₁₀aryl(C₁₋₆)alkyl, C₁₋₆alkylcarbonyl, C₂₋₆ carboxyalkyl, cyano, andtrifluoromethoxy.

R¹ is more preferably one of C₁₋₈alkyl such as ethyl, propyl orisopropyl; cycloalkyl, such as cyclohexyl; or C₆₋₁₀aryl, such as phenyl.Most preferred is isopropyl.

Preferred values of R² are C₁₋₈alkyl, C₃₋₈cycloalkyl, especiallyC₃₋₆cycloalkyl, C₁₋₈alkoxy, C₂₋₈alkenyl, C₂₋₈alkynyl C₆₋₁₄aryl,especially C₆₋₁₀aryl, C₆₋₁₀ar(C₁₋₆)alkyl or C₁₋₆alk(C₆₋₁₀)aryl, wherethe ring portion of any of said aryl, aralkyl, or alkaryl can beoptionally substituted with any of the substituents as described for R¹above.

R² is more preferably C₁₋₄alkyl, such as methyl, ethyl, propyl, orbutyl; or C₁₋₄alkoxy, such as methoxy, or ethoxy. Most preferred aremethyl, ethyl and propyl, and butyl.

With respect to R³, a variety of ester functionalities can be employedat this position. Preferred values are C₁₋₈alkyl, C₃₋₈cycloalkyl,especially C₄₋₇cycloalkyl, C₂₋₈alkenyl, C₂₋₈alkynyl, C₆₋₁₄aryl,especially C₆₋₁₀aryl, C₆₋₁₀ar(C₁₋₆) alkyl or C₁₋₆alk(C₆₋₁₀)aryl, any ofwhich can be optionally substituted. Substituents that can be optionallypresent on R³ include one or more, preferably one or two, of thesubstituents as described for R¹ above.

R³ is more preferably C₁₋₄alkyl, C₆₋₁₀aryl or C₆₋₁₀ ar(C₁₋₆)alkyl. Mostpreferred are methyl, ethyl, tert-butyl and benzyl.

R⁴ is preferably C₆₋₁₀ aryl, preferably phenyl, or a heteroaryl groupselected from the group consisting of thienyl, benzo[b]thienyl, furyl,pyranyl, isobenzofuranyl, benzoxazolyl, 2H-pyrrolyl, pyrrolyl,imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl,indolizinyl, isoindolyl, 3H-indolyl, indolyl, indazolyl, purinyl,4H-quinolizinyl, isoquinolyl, quinolyl, ortriazolyl. The phenyl orheteroaryl group can be optionally substituted by one or two of thesubstituents as described for R¹ above. Most preferred are phenyl, andphenyl substituted by halogen, C₁₋₆alkyl, C₁₋₆alkoxy, carboxy, amino,C₁₋₆alkylamino and/or di(C₁₋₆)alkylamino.

R⁵ and R⁶ are independently one of alkyl, aralkyl or alkaryl; or R⁵ andR⁶ when taken together with the nitrogen atom to which they are attachedform a 5- to 7-membered heterocyclic ring, which can be optionallysubstituted, and which optionally can include an additional oxygen ornitrogen atom. Optional substituents are those listed above for R¹.

R⁵ and R⁶ are preferably C₁₋₆alkyl, C₆₋₁₀ar(C₁₋₆)alkyl orC₁₋₆alk(C₆₋₁₀)aryl or together with the nitrogen atom to which they areattached form a 5- to 7-membered heterocycle which can be optionallysubstituted, and which optionally can include an additional oxygen ornitrogen atom. Most preferred values for NR⁵R⁶ are dimethylamino,diethylamino, pyrrolidino, piperidino, morpholino, oxazolidinone, andoxazolidinone substituted by halogen, C₁₋₆alkyl, C₆₋₁₀ ar(C₁₋₆)alkyl,C₁₋₆alkoxy, carboxy, and/or amino.

R⁷ is preferably C₁₋₈alkyl, C₃₋₈cycloalkyl, C₆₋₁₀aryl,C₆₋₁₀ar(C₁₋₆)alkyl, C₁₋₆alk(C₆₋₁₀)aryl, any of which can be optionallysubstituted. Substituents that can be optionally present on either orboth of the ring or chain portions of R⁷ include one or more, preferablyone or two, of the substituents as described for R¹ above. Preferably,R⁷ together with the sulfur atom to which it is attached is cysteine ora derivative of cysteine such as N-acetyl cysteine, glutathione, and thelike.

Scheme 1 is a general scheme for forming lactacystin andclasto-lactacystin-β-lactone analogs from substituted oxazoline startingmaterials.

The starting oxazoline I, which may be of either the cis (Ia) or trans(Ib) configuration, is deprotonated with a strong base to form theenolate. Examples of bases suitable for use in this reaction are organicbases, including hindered amide bases such as lithium diisopropylamide(LDA), lithium tetramethylpiperidide (LiTMP), lithium, sodium orpotassium hexamethyldisilazide (LiHMDS, NaHMDS, KHMDS), or the like; orhindered alkyllithium reagents, such as sec-butyllithium,tert-butyllithium, or the like. The reaction is preferably conducted atreduced temperature in an ethereal solvent, such as diethyl ether,tetrahydrofuran (THF), or dimethoxyethane (DME). Reaction temperaturespreferably range from about −100° C. to about −30° C., more preferablyfrom −85° C. to −50° C., and most preferably from −85° C. to −75° C. Thereaction temperature is important in determining the stereochemicaloutcome of the subsequent addition to the aldehyde, with lowertemperatures providing better selectivity.

The deprotonation step is followed by transmetallating said enolate witha metal selected from the group consisting of titanium, aluminum, tin,zinc, magnesium and boron. Preferred reagents for this step includetitanium or aluminum Lewis acids, for example Me₂AlCl or (i-PrO)₃TiCl ora mixture of the two. Preferably, between one and three molarequivalents of the Lewis acid are used, more preferably between two andthree equivalents, and most preferably about 2.2-2.3 equivalents.Subsequent treatment of the enolate with a formyl amide (XIV) affordsthe adduct II. Excess aldehyde is washed away with sodium bisulfitesolution, and the crude material is carried forward to the next stepwithout further purification. The use of 2.2-2.3 equivalents of Me₂AlClresults in selective formation of the (6S)-product (lactacystinnumbering), in a ratio generally better than about 10:1, whereas the useof 1 equivalent of Me₂AlCl results in selective formation of the(6R)-product, in a ratio of about 5:1.

Catalytic hydrogenolysis of the adduct II, as a mixture of (6S)- and(6R)-epimers, affords the desired γ-lactam (IV), sometimes as a mixturewith the aminodiol III:

Useful catalysts for this reaction include palladium black, palladium onactivated carbon, palladium hydroxide on carbon, or the like. Organicsolvents suitable for use in this reaction include lower alkanols suchas methanol, ethanol, or isopropanol, lower alkanoates such as ethylacetate, lower alkanoic acids such as acetic acid, or mixtures thereof.The reaction is conducted under an atmosphere of hydrogen, at pressuresranging from about 15 to about 100 p.s.i., more preferably from about 30to about 50 p.s.i. Alternatively, transfer hydrogenation procedures (R.A. W. Johnstone et al., Chem. Rev. 85:129 (1985)) may be used, in whichthe adduct II is treated at atmospheric pressure with a catalyst and ahydrogen donor.

Upon heating of the crude product mixture, the aminodiol III isconverted to the γ-lactam IV, which can then be isolated inapproximately 60-75% overall yield from II. The heating step isconveniently carried out by first filtering off the catalyst used in thehydrogenation step and then heating the filtrate to reflux. When noaminodiol III is present in the crude product mixture, the heating stepis omitted. Ester saponification, followed by cyclization, affords theβ-lactone VII in 40-90% yield, and generally in greater than 60% yield.Cyclization can be effected with coupling reagents known in the art,including aryl sulfonyl chlorides,benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate(BOP reagent), O-(1H-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate (TBTU), alkyl, aryl or alkenyl chloroformates, and thelike. Isopropenyl chloroformate is a preferred reagent for this step,since all byproducts are volatile and chromatographic purification ofthe product is not necessary.

clasto-Lactacystin β-lactone can be converted to lactacystin bytreatment of the β-lactone with N-acetylcysteine according to thereported procedure (Corey et al., Tetrahedron Lett. 34:6977 (1993)).Reactions of the β-lactone VII with other thiols proceed analogously.Alternatively, lactacystin analogs are prepared by coupling thecarboxylic acid intermediate V with a thiol to form the correspondingthiolester VI. The method of this invention is therefore useful forsynthesis of both lactacystin and clasto-lactacystin β-lactone, as wellas analogs thereof.

The enantiomerically-enriched formyl amides XIV employed in the aldolreaction are new. They can be prepared according to a representativereaction sequence such as that depicted in Scheme 2. For purposes of thepresent invention, the term “enantiomerically-enriched” means that oneenantiomer is present in excess relative to the other; that is, oneenantiomer represents greater than 50% of the mixture. The term“stereoselective” is used to mean that a synthesis or reaction stepproduces one enantiomer or diastereomer in excess relative to the otherenantiomer or to other diastereomer(s).

Acylation of the anion of (S)-(−)-4-benzyl-2-oxazolidinone (VIIIa) or(S)-(−)-4-isopropyl-2-oxazolidinone (VIIIb) (where R⁸ is benzyl orisopropyl) affords the acyloxazolidinone IX in greater than 80% yield.Subsequent stereoselective benzyloxymethylation (Evans et al., J. Am.Chem. Soc. 112:8215 (1990)) gives the protected alcohol X in greaterthan 80% yield, provided that the benzyl chloromethyl ether is freshlyprepared (Connor et al., Organic Syntheses 52:16 (1974)). Peroxidemediated hydrolysis affords the acid XI, which is coupled with an amineto provide the amide XII, generally in greater than 50% overall yield.Benzyl group hydrogenolysis, followed by oxidation of the resultantalcohol (XIII) then affords the formyl amide XIV in 80-85% yield.Pearlmans catalyst (Pd(OH)₂) is preferably used for the hydrogenolysisstep. The final oxidation step is best accomplished with the periodinanereported by Dess and Martin, J. Org. Chem. 48:4156 (1983) or with2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) free radical, and bufferedhypochlorite in the presence of bromide ion (J. Org. Chem. 50:4888(1985); Org. Synth. Coil. 8:367 (1993)). Other mild oxidants such astetrapropyl-ammonium perruthenate (TPAP) can also be used. The formylamide XIV can be shown to be enantiomerically pure by reducing thealdehyde with sodium borohydride and converting the resultant alcohol tothe corresponding Mosher ester using R-(+)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride (Dale et al., J. Org. Chem. 34:2543 (1969)). ¹HNMR analysis at 300 MHz reveals a single diastereomer. The aldehydesprepared according to Scheme 2 are configurationally stable, showing nosigns of enantiomeric deterioration after one week, when stored at 0° C.The aldehyde is also configurationally stable under the conditions ofthe aldol reaction, and the the adduct II is formed withoutepimerization of the substituent R² at C(7).

The synthetic methods will work with any substituent at R¹ that isstable to strong base and to hydrogenation. Isopropyl is the preferredsubstituent for good proteasome inhibiting activity of the finalproduct.

The invention also relates to a new route to form the oxazoline startingmaterial I. The overall synthesis includes five steps (Scheme 3) andaffords the cis-substituted oxazoline Ia, which is thereafter employedin the method described above. The first step depicted in Scheme 3 isSharpless asymmetric dihydroxylation (Sharpless et al., J. Org. Chem.57:2768 (1992); Kolb et al., Chem. Rev. 94:2483 (1994); Shao andGoodman, J. Org. Chem. 61:2582(1996)) of the alkene XV. If notcommercially available, the alkene XV is prepared by Wittig condensationbetween the aldehyde and carboethoxymethylene triphenylphosphorane (Haleet al., Tetrahedron 50:9181 (1994)). Other olefination procedures arealso known in the art. The dihydroxylation reaction is preferablyconducted with AD-mix-β (Aldrich Chemical Co.) in the presence ofmethane sulfonamide and stereoselectively affords the diol XVIa, aspredicted by the Sharpless face-selection rule. On a large scale, thedihydroxylation reaction is preferably conducted usingN-methylmorpholine-N-oxide (NMO) as the reoxidant in place of K₃Fe(CN)₆present in AD-mix-β. Although proceeding with somewhat lowerenantioselectivity, this procedure allows more concentrated reactionmixtures and greatly simplifies the workup. The enantiomeric purity ofthe product can be enhanced by recrystallization.

In the next step, the diol XVIa is treated with an orthoester underLewis or Brönsted acid catalysis to give a mixed orthoester, which isconverted in situ to the haloester XVIIa by treatment with an acylhalide (Haddad et al., Tetrahedron Lett. 37:4525 (1996)). Although acylhalides, especially acetyl halides are preferred for this reaction,other acid halides such as HCl, HBr, HI, Me₃SiCl, Me₃SiI, Me₃SiBr andthe like may be used. Halogen-containing Lewis acids of the formulaML_(n)X, such as BBr₃, SnCl₄, Ti(OR)₂Cl₂, Ti(OR)₃Cl, and the like canalso be used. In the previous formula, M is a metal selected from thegroup consisting of B, Ti, Sn, Al, Zn, and Mg; L is any suitable ligandfor the metal, preferably an alkoxide or halogen group; n is an integerthat results in a stable complex, and X is a halogen. Preferably acetylbromide is used to produce the haloester XVIIa. Preferably theorthoester employed in this reaction is derived from an aromatic orheteroaromatic carboxylic acid. More preferably, the orthoester isderived from benzoic acid, e.g., trimethyl orthobenzoate. The use ofboron trifluoride etherate as the Lewis acid catalyst in the formationof the mixed orthoester is preferred, but other acids, such as HBr,SnCl₄, TiCl₄, BBr₃, and the like, can also be used.

After workup, the crude halide XVIIa is converted to the azide XVIIIa bytreatment with an alkali metal azide in a polar aprotic organic solvent,such as dimethyl sulfoxide (DMSO) or N,N-dimethyl formamide (DMF).Catalytic hydrogenation of the azide XVIIIa over a palladium catalyst inethyl acetate proceeds with concomitant migration of the aroyl group(Wang et al., J. Org. Chem. 59:5014 (1994)) to afford the hydroxyamideXIXa.

Treatment of XIXa with thionyl chloride in methylene chloride effectsring closure with inversion of configuration at the hydroxyl-substitutedcarbon atom to produce the cis-substituted oxazoline starting materialIa. Other reagents suitable for use in this reaction include sulfurylchloride, phosphorous trichloride, phosphorous oxychloride, and(methoxycarbonylsulfamoyl)-triethylammonium hydroxide, inner salt(Burgess reagent). Treatment of XIXa under Mitsunobu conditions(Mitsunobu, Synthesis:1 (1981) will also effect a ring closure. Theoxazoline ring oxygen atom is destined to become the C(9)-hydroxyl groupin the final products VI and VII. Under equilibrating conditions (sodiummethoxide, methanol), the cis-oxazoline (Ia) is converted to thetrans-oxazoline (Ib) by inversion of configuration of the estersubstituent, with the configuration of the R¹ substituent remainingfixed. The cis- and trans-oxazolines can both be used in the methoddepicted in Scheme 1, with equivalent results.

In an alternative route to form the oxazoline starting material I,p-toluenesulfonic acid (p-TsOH) is used to effect ring closure (Scheme4). In this case, ring closure proceeds with retention of configurationat the hydroxyl-substituted carbon atom to afford the trans-oxazoline(Ib). In order to obtain the proper stereochemistry at C(9) of the finalproduct, the chiral ligand employed in the dihydroxylation reaction mustbe selected so as to provide the opposite face selectivity from thatdepicted in Scheme 3. For example, AD-mix-α is used in place ofAD-mix-β. All other steps in the sequence proceed analogously to thosedescribed for the synthesis of the cis-oxazoline Ia.

Compounds

Many of the compounds described above are novel compounds; the novelcompounds are also claimed.

Fourth, fifth and sixth aspects of the invention relate to lactacystinanalogs that can be made by the synthetic routes described herein; topharmaceutical compositions including such compounds; and to methods oftreating a subject having a condition mediated by proteins processed bythe proteasome by administering to a subject an effective amount of apharmaceutical composition disclosed herein. These methods includetreatments for Alzheimers disease, cachexia, cancer, inflammation (e.g.,inflammatory responses associated with allergies, bone marrow or solidorgan transplantation, or disease states, including but not limited toarthritis, multiple sclerosis, inflammatory bowel disease and parasiticdiseases such as malaria), psoriasis, restenosis, stroke, and myocardialinfarction.

The compounds of Formulae VI and VII disclosed herein are highlyselective for the proteasome, and do not inhibit other proteases such astrypsin, α-chymotrypsin, calpain I, calpain II, papain, and cathepsin B.

As disclosed by Fenteany et al. (WO 96/32105), hereby incorporated byreference in its entirety, lactacystin, clasto-lactacystin β-lactone,and analogs thereof possess biological activity as inhibitors of theproteasome. They can be used to treat conditions mediated directly bythe function of the proteasome, such as muscle wasting, or mediatedindirectly via proteins which are processed by the proteasome, such asthe transcription factor NF-κB. The compounds prepared by the methods ofthis invention can also be used to determine whether a cellular,developmental, or physiological process or output is regulated by theproteolytic activity of the proteasome.

Those compounds that possess unexpected proteasome function-inhibitingactivity are compounds of Formulae VI and VII:

or a salt thereof, wherein:

R¹ is C₁₋₁₂alkyl, C₃₋₈cycloalkyl, C₂₋₈alkenyl, C₂₋₈alkynyl, C₆₋₁₄aryl,C₆₋₁₀ ar(C₁₋₆)alkyl or C₁₋₆alk(C₆₋₁₀)aryl;

R² is C₂₋₆alkyl; and

R⁷ is C₁₋₈ alkyl, C₃₋₈cycloalkyl, C₆₋₁₀aryl, C₆₋₁₀ar(C₁₋₆)alkyl,C₁₋₆alk(C₆₋₁₀)aryl, any of which can be optionally substituted.Substituents that can be optionally present on either or both of thering or chain portions of R⁷ include one or more, preferably one or two,of the substituents as described for R¹ above.

Preferred compounds are those where R¹ is C₁₋₄ alkyl, more preferablyisopropyl. R² is preferably ethyl, n-propyl, n-butyl or isobutyl.Preferably, R⁷ together with the sulfur atom to which it is attached iscysteine or a derivative of cysteine such as N-acetyl cysteine,glutathione, and the like.

A seventh aspect of the present invention is directed toenantiomerically-enriched formyl amides of Formula XIV:

or salts thereof, wherein

R² is C₁₋₈alkyl, C₃₋₈cycloalkyl, C₂₋₈alkenyl, C₂₋₈alkynyl, C₆₋₁₄aryl,C₆₋₁₀ ar(C₁₋₆)alkyl or C₁₋₆alk(C₆₋₁₀)aryl; and

R⁵ and R⁶ are independently C₁₋₆alkyl, C₆₋₁₀ar(C₁₋₆)alkyl orC₁₋₆alk(C₆₋₁₀)aryl, or together with the nitrogen atom to which they areattached form a 5- to 7-membered heterocycle which can be optionallysubstituted, and which optionally can include an additional oxygen ornitrogen atom.

Preferred compounds are those where R² is C₂₋₆alkyl.

An eighth aspect of the present invention is directed to compounds ofFormulae II and III:

or salts thereof wherein

R¹ is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkaryl, aralkyl, wherethe ring portion of any of said aryl, aralkyl, or alkaryl can beoptionally substituted;

R² is alkyl, cycloalkyl, aryl, alkaryl, aralkyl, alkoxy, hydroxy,alkoxyalkyl, or amido, where the ring portion of any of said aryl,aralkyl, or alkaryl can be optionally substituted;

R³ is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkaryl, any of whichcan be optionally substituted;

R⁴ is optionally substituted aryl or optionally substituted heteroaryl;and

R⁵ and R⁶ are independently one of alkyl or alkaryl; or R¹ and R⁶ whentaken together with the nitrogen atom to which they are attached form a5- to 7-membered heterocyclic ring, which can be optionally substituted,and which optionally include an additional oxygen or nitrogen atom. Mostpreferred values for NR⁵R⁶ are dimethylamino, diethylamino, pyrrolidino,piperidino, morpholino, oxazolidinone, and oxazolidinone substituted byhalogen, C₁₋₆alkyl, C₆₋₁₀ ar(C₁₋₆alkyl, C₁₋₆alkoxy, carboxy, and/oramino.

Preferred compounds of Formulae II and III are those wherein:

R¹ is C₁₋₁₂alkyl, C₃₋₈cycloalkyl, C₂₋₈alkenyl, C₂₋₈alkynyl, C₆₋₁₄aryl,C₆₋₁₀ ar(C₁₋₆)alkyl or C₁₋₆alk(C₆₋₁₀)aryl, where the ring portion of anyof said aryl, aralkyl, or alkaryl can be optionally substituted;

R² is C₁₋₈alkyl, C₃₋₈cycloalkyl, C₂₋₈alkenyl, C₂₋₈alkynyl, C₆₋₁₄aryl,C₆₋₁₀ ar(C₁₋₆)alkyl or C₁₋₆alk(C₆₋₁₀)aryl, where the ring portion of anyof said aryl, aralkyl, or alkaryl can be optionally substituted;

R³ is C₁₋₈alkyl, C₃₋₈cycloalkyl, C₂₋₈alkenyl, C₂₋₈alkynyl, C₆₋₄aryl,C₆₋₁₀ ar(C₁₋₆)alkyl or C₁₋₆alk(C₆₋₁₀)aryl, any of which can beoptionally substituted;

R⁴ is optionally substituted C₆₋₁₀aryl, or an optionally substitutedheteroaryl group selected from the group consisting of thienyl,benzo[β]thienyl, furyl, pyranyl, isobenzofuranyl, benzoxazolyl,2H-pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl,pyrimidinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, indolyl,indazolyl, purinyl, 4H-quinolizinyl, isoquinolyl, quinolyl, ortriazolyl; and

R⁵ and R⁶ are independently C₁₋₆alkyl, C₆₋₁₀ ar(C₁₋₆)alkyl orC₁₋₆alk(C₆₋₁₀)aryl, or together with the nitrogen atom to which they areattached form a 5- to 7-membered heterocycle which can be optionallysubstituted, and which optionally can include an additional oxygen ornitrogen atom. Most preferred values for NR⁵R⁶ are dimethylamino,diethylamino, pyrrolidino, piperidino, morpholino, oxazolidinone, andoxazolidinone substituted by halogen, C₁₋₆alkyl, C₆₋₁₀ar(C₁₋₆)alkyl,C₁₋₆ alkoxy, carboxy, and/or amino.

A ninth aspect of the present invention is directed to compounds ofFormulae XVIIa, XVIIb, XVIIIa, XVIIIb, XIXa or XIXb:

or salts thereof, wherein

R¹ is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkaryl, aralkyl, wherethe ring portion of any of said aryl, aralkyl, or alkaryl can beoptionally substituted;

R³ is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, alkaryl, any of whichcan be optionally substituted; and

R⁴ is optionally substituted aryl or optionally substituted heteroaryl.

Preferred compounds of Formulae XVII, XVIII or XIX are those wherein

R¹ is C₁₋₁₂alkyl, C₃₋₈cycloalkyl, C₂₋₈alkenyl, C₂₋₈alkynyl C₆₋₁₄aryl,C₆₋₁₀ ar(C₁₋₆)alkyl or C₁₋₆alk(C₆₋₁₀)aryl, where the ring portion of anyof said aryl, aralkyl, or alkaryl can be optionally substituted;

R³ is C₁₋₈alkyl, C₃₋₈cycloalkyl, C₂₋₈alkenyl, C₂₋₈alkynyl, C₆₋₁₄aryl,C₆₋₁₀ ar(C₁₋₆)alkyl or C₁₋₆alk(C₆₋₁₀)aryl, any of which can beoptionally substituted; and

R⁴ is optionally substituted C₆₋₁₀aryl, or an optionally substitutedheteroaryl group selected from the group consisting of thienyl,benzo[b]thienyl, furyl, pyranyl, isobenzofuranyl, benzoxazolyl,2H-pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl,pyrimidinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, indolyl,indazolyl, purinyl, 4H-quinolizinyl, isoquinolyl, quinolyl, ortriazolyl.

Definitions

The term “alkyl” as employed herein includes both straight and branchedchain radicals of up to 12 carbons, preferably 1-8 carbons, such asmethyl, ethyl, propyl, isopropyl, butyl, t-butyl, isobutyl, pentyl,hexyl, isohexyl, 1-ethylpropyl, heptyl, 4,4-dimethylpentyl, octyl,2,2,4-trimethylpentyl, nonyl, decyl, undecyl and dodecyl.

The term “substituted alkyl” as employed herein, includes alkyl groupsas defined above that have one, two or three halo, hydroxy, nitro,trifluoromethyl, halogen, C₁₋₆alkyl, C₆₋₁₀aryl, C₁₋₆alkoxy,C₁₋₆aminoalkyl, C₁₋₆aminoalkoxy, amino, C₂₋₆alkoxycarbonyl, carboxy,C₁₋₆hydroxyalkyl, C₂₋₆hydroxyalkoxy, C₁₋₆ alkylsulfonyl,C₆₋₁₀arylsulfonyl, C₁₋₆alkylsulfinyl, C₁₋₆alkylsulfonamido, C₆₋₁₀arylsulfonamido, C₆₋₁₀ar(C₁₋₆)alkylsulfonamido, C₁₋₆alkyl,C₁₋₆hydroxyalkyl, C₆₋₁₀ aryl, C₆₋₁₀aryl(C₁₋₆)alkyl, C₁₋₆alkylcarbonyl,C₂₋₆carboxyalkyl, cyano, and trifluoromethoxy and/or carboxysubstituents.

The term “cycloalkyl” as employed herein includes saturated cyclichydrocarbon groups containing 3 to 12 carbons, preferably 3 to 8carbons, which include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, cyclooctyl, cyclodecyl and cyclododecyl, any of whichgroups may be substituted with substituents such as halogen, C₁₋₆alkyl,C₁₋₆alkoxy and/or hydroxy group.

The term “heteroaryl” as employed herein refers to groups having 5 to 14ring atoms, preferably 5, 6, 9 or 10 ring atoms; 6, 10 or 14 π electronsshared in a cyclic array; and containing carbon atoms and 1, 2 or 3oxygen, nitrogen or sulfur heteroatoms (where examples of heteroarylgroups are: thienyl, benzo[b]thienyl, naphtho[2,3-b]thienyl,thianthrenyl, furyl, pyranyl, isobenzofuranyl, benzoxazolyl, chromenyl,xanthenyl, phenoxathiinyl, 2H-pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl,pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolizinyl, isoindolyl,3H-indolyl, indolyl, indazolyl, purinyl, 4H-quinolizinyl, isoquinolyl,quinolyl, phthalazinyl, naphthyridinyl, tetrazolyl, quinazolinyl,cinnolinyl, pteridinyl, 4αH-carbazolyl, carbazolyl, β-carbolinyl,phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl, phenazinyl,isothiazolyl, phenothiazinyl, isoxazolyl, furazanyl and phenoxazinylgroups).

The term “aryl” as employed herein by itself or as part of another grouprefers to monocyclic or bicyclic aromatic groups containing from 6 to 12carbons in the ring portion, preferably 6-10 carbons in the ringportion, such as phenyl, naphthyl or tetrahydronaphthyl.

The term “aralkyl” or “arylalkyl” as employed herein by itself or aspart of another group refers to C₁₋₆alkyl groups as discussed abovehaving an aryl substituent, such as benzyl, phenylethyl or2-naphthylmethyl.

The term “alkaryl” or “alkylaryl” as employed herein by itself or aspart of another group refers to an aryl group as discussed above havinga C₁₋₆ alkyl substituent, such as toluyl, ethylphenyl, ormethylnaphthyl.

The term “optionally substituted” when used with respect to aryl,aralkyl, alkaryl or 5-, 6-, 9- or 10-membered heteroaryl groups meansthat the ring portion of said groups can be optionally substituted byone or two substituents independently selected from C₁₋₆alkyl,C₃₋₈cycloalkyl, C₁₋₆alkyl(C₃₋₈)cycloalkyl, C₂₋₈alkenyl, C₂₋₈alkynyl,cyano, amino, C₁₋₆alkylamino, di(C₁₋₆)alkylamino, benzylamino,dibenzylamino, nitro, carboxy, carbo(C₁₋₆)alkoxy, trifluoromethyl,halogen, C₁₋₆alkoxy, C₆₋₁₀aryl, C₆₋₁₀aryl(C₁₋₆)alkyl,C₆₋₁₀aryl(C₁₋₆)alkoxy, hydroxy, C₁₋₆alkylthio, C₁₋₆alkylsulfinyl,C₁₋₆alkylsulfonyl, C₆₋₁₀arylthio, C₆₋₁₀arylsulfinyl, C₆₋₁₀arylsulfonyl,C₆₋₁₀aryl, C₁₋₆alkyl(C₆₋₁₀)aryl, and halo(C₆₋₁₀)aryl.

The term “alkoxy” refers to the above alkyl groups linked to oxygen.

The term “halogen” or “halo” as employed herein by itself or as part ofanother group refers to chlorine, bromine, fluorine or iodine.

The term “amido” as employed herein refers to formylamino,alkylcarbonylamino or arylcarbonylamino.

Uses

Pharmacological data for clasto-lactacystin β-lactone analogs preparedby the methods of this invention are provided in Table 1. Thesecompounds are all irreversible inactivators of the 20S proteasome,acylating the N-terminal threonine residue of the X/MB1 subunit. Thevalue K_(obs)/[I] is a measure of the rate of enzyme inactivation.Several compounds show improved activity, i.e., more rapid rates ofinactivation, when compared to clasto-lactacystin β-lactone itself(2).The compound that is most potent in the enzyme assay is the 7-methoxyderivative 3f. However, when assayed in cell culture, 3f is less potentthan 2.

The lactone ring is subject to nucleophilic attack not only by thethreonine residue of the proteasome X/MB1 subunit, but also by water.Hydrolysis results in formation of the hydroxy acid V, which is notactive as an inhibitor of the proteasome. Relative potency in cellculture is a composite of many factors, including enzyme potency, cellpenetration, and hydrolysis rate. Although more potent than 2 againstthe enzyme, 3f is also more rapidly hydrolyzed, resulting in much weakeractivity in cell culture. By contrast, the analogs 3a-3d showunexpectedly improved potency not only in the enzyme assay, but also incell culture.

The disclosed compounds are used to treat conditions mediated directlyby the proteolytic function of the proteasome such as muscle wasting, ormediated indirectly via proteins which are processed by the proteasomesuch as NF-κB. The proteasome participates in the rapid elimination andpost-translational processing of proteins involved in cellularregulation (e.g., cell cycle, gene transcription, and metabolicpathways), intercellular communication, and the immune response (e.g.,antigen presentation). Specific examples include β-amyloid protein andregulatory proteins such as cyclins and transcription factor NF-κB.Treating as used herein includes reversing, reducing, or arresting thesymptoms, clinical signs, and underlying pathology of a condition in amanner to improve or stabilize the subject's condition.

Other embodiments of the invention relate to cachexia and muscle-wastingdiseases. The proteasome degrades many proteins in maturingreticulocytes and growing fibroblasts. In cells deprived of insulin orserum, the rate of proteolysis nearly doubles.

Inhibiting the proteasome reduces proteolysis, thereby reducing bothmuscle protein loss and the nitrogenous load on kidneys or liver.Proteasome inhibitors are useful for treating conditions such as cancer,chronic infectious diseases, fever, muscle disuse (atrophy) anddenervation, nerve injury, fasting, renal failure associated withacidosis, and hepatic failure. See, e.g., Goldberg, U.S. Pat. No.5,340,736 (1994).

Embodiments of the invention therefore encompass methods for reducingthe rate of muscle protein degradation in a cell, and reducing the rateof intracellular protein degradation. Each of these methods includes thestep of contacting a cell (in vivo or in vitro, e.g., a muscle in asubject) with an effective amount of a compound (e.g., pharmaceuticalcomposition) of a formula disclosed herein.

Proteasome inhibitors block processing of ubiquitinated NF-κB in vitroand in vivo. Proteasome inhibitors also block IκB-α degradation andNF-κB activation. (Palombella, et al.; and Traenckner, et al., EMBO J.13:5433-5441 (1994)). One embodiment of the invention is a method forinhibiting IκB-α degradation, including contacting the cell with acompound of a formula described herein. A further embodiment is a methodfor reducing the cellular content of NF-κB in a cell, muscle, organ, orsubject, including contacting the cell, muscle, organ, or subject with acompound of a formula described herein. Additional embodiments encompassmethods for treating inflammatory responses associated with allergies,bone marrow or solid organ transplantation, or disease states, includingbut not limited to arthritis, inflammatory bowel disease, asthma, andmultiple sclerosis by administering a compound of a formula disclosedherein. A preferred embodiment of the invention is directed to treatingasthma by administering a compound of Formula VI or Formula VII, mostpreferably compound 3b.

Proteasome inhibitors are also useful for treatment of ischemic orreperfusion injury, particularly for preventing or reducing the size ofinfarct after vascular occlusion such as occurs during a stroke or heartattack, as described in Brand, U.S. Pat. No. 6,271,199 (2001).Proteasome inhibitors also block proteasome-dependent transformation ofprotozoan parasites (Gonzalez et al., J. Exp. Med. 184:1909 (1996).Further embodiments of the invention therefore encompass methods fortreating an infarct or a protozoan parasitic disease by administering acompound of a formula disclosed herein. In a preferred aspect of theinvention, a compound of Formula VI or Formula VII is administered toprevent or reduce the size of the infarct after vascular occlusion. Saidcompounds can be administered from about 0 to about 10 hours from theoccurrence of a stroke in order to treat or reduce neuronal lossfollowing an ischemic event. Compound 3b is the most preferred compoundin this aspect of the invention.

Proteasome inhibitors also block degradation of cell cycle regulatoryproteins, such as cyclins and cyclin-dependent kinase inhibitors, andtumor suppressor proteins, such as p53. Other embodiments of theinvention therefore encompass methods for blocking the cell cycle andfor treating cell proliferative diseases such as cancer, psoriasis, andrestenosis with a compound of a formula described herein.

The term “inhibitor” is meant to describe a compound that blocks orreduces the activity of an enzyme (e.g., the proteasome, or the X/MB1subunit of the 20S proteasome). An inhibitor may act with competitive,uncompetitive, or noncompetitive inhibition. An inhibitor may bindreversibly or irreversibly, and therefore the term includes compoundswhich are suicide substrates of an enzyme. An inhibitor may modify oneor more sites on or near the active site of the enzyme, or it may causea conformational change elsewhere on the enzyme.

Amounts and regimens for the administration of proteasome inhibitors andcompositions of the invention can be determined readily by those withordinary skill in the clinical art. Generally, the dosage of thecomposition of the invention will vary depending upon considerationssuch as: type of composition employed; age; health; medical conditionsbeing treated; kind of concurrent treatment, if any, frequency oftreatment and the nature of the effect desired; extent of tissue damage;gender; duration of the symptoms; and, counter indications, if any, andother variables to be adjusted by the individual physician. A desireddosage can be administered in one or more applications to obtain thedesired results. Pharmaceutical compositions containing the proteasomeinhibitors of the invention can be provided in unit dosage forms.

Compositions within the scope of this invention include all compositionswherein the compounds of the present invention are contained in anamount which is effective to achieve its intended purpose. Whileindividual needs vary, determination of optimal ranges of effectiveamounts of each component is within the skill of the art. Typically, thecompounds may be administered to mammals, e.g. humans, orally at a doseof 0.0025 to 50 mg/kg, or an equivalent amount of the pharmaceuticallyacceptable salt thereof, per day of the body weight of the mammal beingtreated for a proteosome-mediated condition such as a stroke or asthma.For intramuscular injection, the dose is generally about one-half of theoral dose.

In the method of prevention or reduction of infarct size the compoundcan be administered by intravenous injection at a dose of about 0.01 toabout 10 mg/kg, preferably about 0.025 to about 1 mg/kg.

The unit oral dose may comprise from about 0.01 to about 50 mg,preferably about 0.1 to about 10 mg of the compound. The unit dose maybe administered one or more times daily as one or more tablets eachcontaining from about 0.1 to about 10, conveniently about 0.25 to 50 mgof the compound or its solvates. For use in treating stroke, it ispreferred that a single dosage be administered, 0 to about 10 hourspost-event, preferably 0 to about 6 hours post-event.

The following examples are illustrative, but not limiting, of the methodand compositions of the present invention. Other suitable modificationsand adaptations of the variety of conditions and parameters normallyencountered and obvious to those skilled in the art are within thespirit and scope of the invention.

The preparation of formyl amides XIV according to the synthetic schemedepicted in scheme 2 as exemplified in Examples 1-6.

EXAMPLE 1 Acyl Oxazolidinones (IX)

a. Acyl oxazolidinone IXb (R²=n-Pr; R⁸=CH₂Ph): A cooled (−78° C.)solution of (S)-(−)-4-benzyl-2-oxazolidinone (4.0 g, 22.6 mmol) in 75 mLanhydrous THF was treated with a 2.5 M solution of n-BuLi in hexane (9.1mL, 22.6 mmol) over 15 min. After 5 min, neat valeryl chloride (2.95 mL,24.9 mmol) was added dropwise and the mixture was stirred for another 45min. at −78° C. The mixture was then allowed to reach room temperature,stirred for another 90 min, and then treated with 50 mL saturated NH₄Clsolution. Dichloromethane (50 mL) was then added and the organic layerwas washed with brine (2×30 mL), dried over MgSO₄ and concentrated invacuo. This afforded 5.94 g (100%) of the desired acyl oxazolidinone IXbas a clear colorless oil. ¹H NMR (300 MHz, CDCl₃) δ 7.36-7.20 (m, 5H),4.71-4.64 (m, 1H), 4.23-4.14 (m, 1H), 3.40 (dd, J=13.3, 3.2 Hz, 1H),3.04-2.84 (m, 2H), 2.77 (dd, J=13.3, 9.6 Hz, 1H), 1.74-1.63 (m, 2H),1.46-1.38 (m, 2H), 0.96 (t, J=7.3 Hz, 3H).

b. Acyl oxazolidinone IXa (R²=Et; R⁸=CH₂Ph): By a procedure analogous tothat described for preparing acyl oxazolidinone IXb, the lithium anionof (S)-(−)-4-benzyl-2-oxazolidinone was treated with butyryl chloride toprovide acyl oxazolidinone IXa in 94% yield. ¹H NMR (300 MHz, CDCl₃) δ7.37-7.20 (m, 5H), 4.68 (ddd, J=13.1, 7.0, 3.4 Hz, 1H), 4.23-4.13 (m,2H), 3.30 (dd, J=13.3, 9.6 Hz, 1H), 3.02-2.82 (m, 2H), 2.77 (dd, J=13.3,9.6Hz, 1H), 1.73 (q, J=7.3 Hz, 2H), 1.01 (t, J=7.3 Hz, 3H).

c. Acyl oxazolidinone IXc (R²=n-Bu; R⁸=CH₂Ph): By a procedure analogousto that described for preparing acyl oxazolidinone IXb, the lithiumanion of (S)-(−)-4-benzyl-2-oxazolidinone was treated with hexanoylchloride to provide acyl oxazolidinone IXc in 96% yield. ¹H NMR (300MHz, CDCl₃) δ 7.36-7.20 (m, 5H), 4.68 (m, 1H), 4.23-4.14 (m, 2H), 3.30(dd, J=13.3, 3.3 Hz, 1H), 3.02-2.83 (m, 2H), 2.76 (dd, J=13.3, 9.6 Hz,1H), 1.70 (m, 2H), 1.43-1.34 (m, 4H), 0.92 (t, J=3.3 Hz, 3H).

d. Acyl oxazolidinone IXd (R²=i-Bu; R⁸=CH₂Ph):

i. 4-Methylvaleryl chloride

4-Methylvaleryl chloride was prepared from commercially available4-methylvaleric acid in the following way: a cold (0° C.) solution of4-methylvaleric acid (1.85 mL, 15.0 mmol) in 50 mL anhydrous CH₂Cl₂containing 10 mL of DMF was treated with 1.95 μL oxalyl chloride (22.5mmol). The mixture was then stirred for 3 h at room temperature,concentrated in vacuo and filtered to affords 1.65 g (100%) of thedesired acid chloride as a colorless liquid.

ii. Acyl oxazolidinone IXd (R²=i-Bu; R⁸=CH₂Ph):

By a procedure analogous to that described for preparing acyloxazolidinone IXb, the lithium anion of (S)-(−)-4-benzyl-2-oxazolidinonewas treated with 4-methylvaleryl chloride to provide acyl oxazolidinoneIXd in 85% yield. ¹H NMR (300 MHz, CDCl₃) δ 7.37-7.20 (m, 5H), 4.70-4.63(m, 1H), 4.23-4.15 (m, 2H), 3.30 (dd, J=13.2, 3.2 Hz, 1H), 2.98-2.90 (m,2H), 2.76 (dd, J=13.3, 9.6 Hz, 1H), 1.68-1.54 (m, 3H), 0.94 (d, J=6.2Hz, 3H).

e. Acyl oxazolidinone IXe (R²=CH₂Ph; R⁸=CH₂Ph): By a procedure analogousto that described for preparing acyl oxazolidinone IXb, the lithiumanion of (S)-(−)-4-benzyl-2-oxazolidinone was treated withhydrocinnamoyl chloride to provide acyl oxazolidinone IXe in 82% yield.¹H NMR (300 MHz, CDCl₃) δ 7.35-7.16 (m, 10H), 4.70-4.63 (m, 1H),4.21-4.14 (m, 2H), 3.38-3.19 (m, 3H), 3.08-2.98 (m, 2H), 2.75 (dd,J=13.4, 9.5 Hz, 1H).

EXAMPLE 2 Acyl Oxazolidinones (X)

a. Acyl oxazolidinone Xb (R²=n-Pr; R⁸=CH₂Ph): A cold (0° C.) solution ofacyl oxazolidinone IXb (5.74 g, 22.0 mmol) in 110 mL anhydrous CH₂Cl₂was treated with 2.52 mL TiCl₄ (23.1 mmol) resulting in the formation ofan abundant precipitate. After 5 min, diisopropylethylamine (4.22 mL,24.2 mmol) was added slowly and the resulting dark brown solution wasstirred at room temperature for 35 min. Benzyl chloromethyl ether (6.0mL, 44.0 mmol) was rapidly added and the mixture was stirred for 5 h atroom temperature. 50 mL CH₂Cl₂ and 75 mL of 10% aqueous NH₄Cl were thenadded, resulting in the formation of yellow gummy material. Afterstirring the suspension vigorously for 10 min, the supernatant wastransferred in a separatory funnel and the gummy residue was taken up in100 mL 1:1 10% aqueous NH₄Cl/CH₂Cl₂. The combined organic layers werethen washed successively with 1N aqueous HCl, saturated NaHCO₃ andbrine, dried over MgSO₄ and concentrated in vacuo. The crude solidmaterial was recrystallized from EtOAc/hexane affording 6.80 g ofdesired acyl oxazolidinone Xb as a white solid in 81% yield. ¹H NMR(300MHz, CDCl₃), δ 7.34-7.18(m, 10H), 4.77-4.69 (m, 1H), 4.55 (s, 2H),4.32-4.23 (m, 1H), 4.21-4.10 (m, 2H), 3.80 (t, J=9.0 Hz, 1H), 3.65 (dd,J=9.0, 5.0 Hz, 1H), 3.23 (dd, J=13.5, 3.3 Hz, 1H), 2.69 (dd, J=13.5, 9.3Hz, 1H), 1.74-1.64 (m, 1H), 1.54-1.44 (m, 1H), 1.40-1.28 (m, 2H), 0.91(t, J=7.3 Hz, 3H). LRMS (FAB) m/e 382 (M+H⁺)

b. Acyl oxazolidinone Xa (R²=Et; R⁸=CH₂Ph): By a procedure analogous tothat described for preparing acyl oxazolidinone Xb, acyl oxazolidinoneXa was obtained in 80% yield. ¹H NMR (300 MHz, CDCl₃) δ 7.36-7.18 (m,10H), 4.55 (s, 2H), 4.21-4.11 (m, 3H), 3.81 (t, J=9.0 Hz, 1H), 3.66 (dd,J=9.0, 5.0 Hz, 1H), 3.23 (dd, J=13.5, 3.2 Hz, 1H), 2.70 (dd, J=13.5, 9.3Hz, 1H), 1.78-1.57 (m, 2H), 0.94 (t, J=7.5 Hz, 3H).

c. Acyl oxazolidinone Xc (R²=n-Bu; R⁸=CH₂Ph): By a procedure analogousto that described for preparing acyl oxazolidinone Xb, acyloxazolidinone Xc was obtained in 91% yield. ¹H NMR (300 MHz, CDCl₃) δ7.38-7.17 (m, 10H), 4.72 (m, 1H), 4.54 (s, 2H), 4.27-4.10 (m, 2H), 3.79(t, J=8.7 Hz, 1H), 3.65 (dd, J=9.1, 5.0 Hz, 1H), 3.23 (dd, J=13.5, 3.3Hz, 1H), 2.68 (dd, J=13.5, 9.3 Hz, 1H), 1.75-1.68 (m, 1H), 1.31-1.26 (m,4H), 0.87 (t, J=6.8 Hz, 3H).

d. Acyl oxazolidinone Xd (R²=i-Bu; R⁸=CH₂Ph): By a procedure analogousto that described for preparing acyl oxazolidinone Xb, acyloxazolidinone Xd was obtained in 98% yield. ¹H NMR (300 MHz, CDCl₃) δ7.38-7.17 (m, 10H), 4.75-4.67 (m, 1H), 4.57 (d, J=12.0 Hz, 1H), 4.51 (d,J=12.0Hz, 1H), 4.41-4.36 (m, 1H), 4.20-4.09 (m, 2H), 3.74(t, J=9.0 Hz,1H), 3.65 (dd, J=9.0, 5.1 Hz, 1H), 3.23 (dd, J=13.5, 3.2 Hz, 1H), 2.63(dd, J=13.5, 9.5 Hz, 1H), 1.74-1.52 (m, 2H), 1.35 (dd, J=13.1, 6.1 Hz,1H), 0.92 (d, J=2.9 Hz, 3H), 0.90 (d, J=2.9 Hz, 3H).

e. Acyl oxazolidinone Xe (R²=CH₂Ph; R⁸=CH₂Ph): By a procedure analogousto that described for preparing acyl oxazolidinone Xb, acyloxazolidinone Xe was obtained in 84% yield. ¹H NMR (300 MHz, CDCl₃) δ7.38-7.15 (m, 15H), 4.62-4.50 (m, 4H), 4.03 (dd, J=9.0, 2.7 Hz, 1H),3.93-3.82 (m, 2H), 3.66 (dd, J=9.2, 4.8 Hz, 1H), 3.19 (dd, J=13.5, 3.2Hz, 1H), 2.98 (dd, J=13.4, 8.2 Hz, 1H), 2.88 (dd, J=13.4, 7.3 Hz, 1H),2.68 (dd, J=13.5, 9.3 Hz, 1H).

EXAMPLE 3 Carboxylic Acids (XI)

a. Carboxylic acid Xb (R²=n-Pr): A cold (0° C.) solution of 6.60 g (17.3mmol) of acyl oxazolidinone Xb in 320 mL THF/H₂O was treatedsuccessively with 6.95 mL 35% aqueous H₂O₂ and a solution of lithiumhydroxide monohydrate (1.46 g, 34.6 mmol) in 20 mL H₂O. The mixture wasstirred for 16 h at 0° C. and then treated carefully first with asolution Na₂SO₃ (10.5 g) in 55 mL H₂O and then with a solution of NaHCO₃(4.35 g) in 100 mL H₂O. The mixture was stirred for 30 min at roomtemperature and concentrated in vacuo to remove the THF. The resultingaqueous mixture was then washed with CH₂Cl₂ (4×75 mL), cooled to 0° C.,acidified with 6N aqueous HCl and extracted with CH₂Cl₂ (1×200 mL and3×100 mL). The combined organic layers were then dried over MgSO₄ andconcentrated in vacuo affording 3.47 g (90%) of desired acid XIb as aclear colorless oil. ¹H NMR (300 MHz, CDCl₃) δ 7.38-7.26 (m, 5H), 4.55(s, 2H), 3.67 (m, 1H), 3.57 (dd, J=9.2, 5.2 Hz, 1H), 2.75 (m, 1H),1.72-1.31 (m, 4H), 0.93 (t, J=7.2 Hz, 3H). LRMS (FAB) m/e 223 (M+H⁺)

b. Carboxylic acid XIa (R²=Et): By a procedure analogous to thatdescribed for preparing acyl oxazolidinone XIb, acyl oxazolidinone XIawas obtained in 48% yield. ¹H NMR (300 MHz, CDCl₃) δ 7.36-7.27 (m, 5H),4.55 (s, 2H), 3.68 (dd, J=9.2, 7.9 Hz, 1H), 3.59 (dd, J=9.2, 5.4 Hz,1H), 2.68-2.65 (m, 1H), 1.71-1.62 (m, 2H), 0.97 (t, J=7.5 Hz, 3H).

c. Carboxylic acid XIc (R²=n-Bu): By a procedure analogous to thatdescribed for preparing acyl oxazolidinone XIb, acyl oxazolidinone XIcwas obtained in 96% yield. ¹H NMR (300 MHz, CDCl₃) δ 7.37-7.28 (m, 5H),4.55 (s, 2H), 3.67 (dd, J=9.1, 8.1 Hz, 1H), 3.57 (dd, J=9.2, 5.3 Hz,1H), 2.72 (m, 1H), 1.67-1.51 (m, 2H), 1.36-1.27 (m, 4H), 0.89 (t, J=6.9Hz, 3H).

d. Carboxylic acid XId (R²=i-Bu): By a procedure analogous to thatdescribed for preparing acyl oxazolidinone XIb, acyl oxazolidinone XIdwas obtained in 80% yield. ¹H NMR (300 MHz, CDCl₃) δ 7.37-7.28 (m, 5H),4.55 (s, 2H), 3.64 (t, J=9.1 Hz, 1H), 3.54 (dd, J=9.1, 5.1 Hz, 1H), 2.81(m, 1H), 1.68-1.54 (m, 2H), 1.36-1.27 (m, 1H), 0.92 (d, J=4.9 Hz, 3H),0.90 (d, J=4.9 Hz, 3H).

e. Carboxylic acid XIe (R²=CH₂Ph): By a procedure analogous to thatdescribed for preparing acyl oxazolidinone XIb, acyl oxazolidinone XIewas obtained in 92% yield. ¹H NMR (300 MHz, CDCl₃) δ 7.38-7.16 (m, 10H),4.53 (d, J=12.1 Hz, 1H), 4.50 (d, J=12.1 Hz, 1H), 3.68-3.57 (m, 2H),3.09-2.85 (m, 3H).

EXAMPLE 4 Diethyl Amides (XII)

a. Diethylamide XIIb (R²=n-Pr; R⁵=R⁶=Et): A cooled solution (0° C.) ofcarboxylic acid XIb (3.40 g, 15.3 mmol) in 1:1 MeCN/CH₂Cl₂ (150 mL),containing diethylamine (2.36 mL, 23.0 mmol) and2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate(TBTU, 5.89 g, 18.4 mmol), was treated with diisopropylethylamine (6.7mL, 38.2 mmol) over 1.5 h (syringe pump). The mixture was thenconcentrated in vacuo and partitioned between ether (200 mL) and H₂O(100 mL). The aqueous layer was extracted with more ether (2×100 mL) andthe combined organic layers were washed with aqueous 1N HCl (3×50 mL),saturated aqueous NaHCO₃ and brine, dried over MgSO₄ and concentrated invacuo. Chromatographic purification (230-400 mesh SiO₂, elution with 1:3AcOEt/hexane) afforded 4.24 g (97%) of diethyl amide XIIb as a clearcolorless oil. ¹H NMR (300 MHz, CDCl₃) δ 7.35-7.23 (m, 5H), 4.52 (d,J=12.0 Hz, 1H), 4.44 (d, J=12.0 Hz, 1H), 3.67 (t, J=8.6 Hz, 1H), 3.51(dd, J=8.7, 5.5 Hz, 1H), 3.46-3.27 (m, 4H), 2.96 (m, 1H), 1.67-1.57 (m,1H), 1.48-1.22 (m, 4H), 1.20-1.10 (m, 6H), 0.90 (t, J=7.2 Hz, 3H). LRMS(FAB) m/e 278 (M+H⁺)

b. Diethylamide XIIa (R²=Et; R⁵=R⁶=Et): By a procedure analogous to thatdescribed for preparing diethylamide XIIb, diethylamide XIIa wasobtained in 73% yield. ¹H NMR (300 MHz, CDCl₃) δ 7.33-7.26 (m, 5H), 4.52(d, J=12.0 Hz, 1H), 4.44 (d, J=12.0 Hz, 1H), 3.68 (t, J=8.6 Hz, 1H),3.53-3.33 (m, 5H), 2.90(m, 1H), 1.75-1.50 (m, 2H), 1.18(t, J=7.1 Hz,3H), 1.13 (t, J=7.1 Hz, 3H), 0.89 (t, J=7.4 Hz, 3H).

c. Diethylamide XIIc (R²=n-Bu; R⁵=R⁶=Et): By a procedure analogous tothat described for preparing diethylamide XIIb, diethylamide XIIc wasobtained in 94% yield. ¹H NMR (300 MHz, CDCl₃) δ 7.35-7.25 (m, 5H), 4.51(d, J=12.0 Hz, 1H), 4.44 (d, J=12.0 Hz, 1H), 3.67 (t, J=8.6 Hz, 1H),3.51 (dd, J=8.8, 5.5 Hz, 1H), 3.46-3.29 (m, 1H), 2.94 (m, 1H), 1.66-1.62(m, 2H), 1.33-1.10 (m, 9H), 0.85 (t, J=7.0 Hz, 3H).

d. Diethylamide XIId (R²=i-Bu; R⁵=R⁶=Et): By a procedure analogous tothat described for preparing diethylamide XIIb, diethylamide XIId wasobtained in 95% yield. ¹H NMR (300 MHz, CDCl₃) δ 7.35-7.23 (m, 5H), 4.51(d, J=12.0 Hz, 1H), 4.44 (d, J=12.0 Hz, 1H), 3.65 (t, J=8.7 Hz, 1H),3.54-3.28 (m, 5H), 3.03 (m, 1H), 1.63-1.49 (m, 2H), 1.33-1.24 (m, 1H),1.18 (t, J=7.1 Hz, 3H), 1.12 (t, J=7.1 Hz, 3H), 0.90 (t, J=6.4 Hz, 3H).

e. Diethylamide XIIe (R²=CH₂Ph; R⁵=R⁶=Et): By a procedure analogous tothat described for preparing diethylamide XIIb, diethylamide XIIe wasobtained in 89% yield. ¹H NMR (300 MHz, CDCl₃) δ 7.35-7.16 (m, 10H),4.53 (d, J=12.1 Hz, 1H), 4.47 (d, J=12.1 Hz, 1H), 3.77 (t, J=8.5 Hz,1H), 3.59 (dd, J=8.8, 5.7 Hz, 1H), 3.40 (m, 1H), 3.22-2.89 (m, 5H), 2.79(dd, J=13.0, 5.1 Hz, 3H), 1.01 (t, J=7.1 Hz, 3H), 0.85 (t, J=7.2 Hz,3H).

EXAMPLE 5 Alcohols (XIII)

a. Alcohol XIIIb (R²=n-Pr; R⁵=R⁶=Et): To a solution of diethylamide XIIb(4.08 g, 14.7 mmol) in 140 mL MeOH was added 20% Pd(OH)₂/C (400 mg) andthe suspension was hydrogenated at atmospheric pressure and roomtemperature for 15 h. Filtration of the catalyst and concentrating thefiltrate in vacuo afforded 2.84 g (100%) of the desired primary alcoholXIIIb. ¹H NMR (300 MHz, CDCl₃) δ 3.74 (br. d, J=4.2 Hz, 1H), 3.61-3.15(m, 5H), 2.71 (m, 1H), 1.69-1.24 (m, 4H), 1.20 (t, J=7.1 Hz, 3H), 1.12(t, J=7.1 Hz, 3H), 0.92 (t, J=7.2 Hz, 3H). LRMS (FAB) m/e 188 (M+H⁺).

b. Alcohol XIIIa (R²=Et; R⁵=R⁶=Et): By a procedure analogous to thatdescribed for preparing alcohol XIIb, alcohol XIIIa was obtained in 100%yield. ¹H NMR (300 MHz, CDCl₃) δ 3.76 (m, 2H), 3.58-3.19 (m, 4H), 2.64(m, 1H), 1.71-1.65 (m, 2H), 1.21 (t, J=7.1 Hz, 3H), 1.13 (t, J=7.1 Hz,3H), 0.96 (t, J=7.4 Hz, 3H).

c. Alcohol XIIIc (R²=n-Bu; R⁵=R⁶=Et): By a procedure analogous to thatdescribed for preparing alcohol XIIIb, alcohol XIIIc was obtained in100% yield. ¹H NMR (300 MHz, CDCl₃) δ 3.76 (d, J=4.5 Hz, 2H), 3.58-3.19(m, 4H), 2.72-2.65 (m, 2H), 1.68-1.55 (m, 2H), 1.40-1.24 (m, 4H), 1.20(t, J=7.1 Hz, 3H), 1.12 (t, J=7.1 Hz, 3H), 0.90 (t, J=6.9Hz, 3H).

d. Alcohol XIIId (R²=i-Bu; R⁵=R⁶=Et): By a procedure analogous to thatdescribed for preparing alcohol XIIIb, alcohol XIIId was obtained in100% yield. ¹H NMR (300 MHz, CDCl₃) δ 3.78-3.68 (m, 2H), 3.57-3.15 (m,4H), 2.81-2.73 (m, 1H), 1.70-1.60(m, 2H), 1.40-1.28(m, 1H), 1.21 (t,J=7.1 Hz, 3H), 1.12 (t, J=7.1 Hz, 3H), 0.92 (m, 6H).

e. Alcohol XIIIe (R²=CH₂Ph; R⁵=R⁶=Et): By a procedure analogous to thatdescribed for preparing alcohol XIIIb, alcohol XIIIe was obtained in100% yield. ¹H NMR (300 MHz, CDCl₃) δ 7.29-7.16 (m, 5H), 3.81-3.71 (m,2H), 3.61-3.50 (m, 1H), 3.15-2.87 (m, 6H), 1.05 (t, J=7.1 Hz, 3H), 0.98(t, J=7.1 Hz, 3H).

EXAMPLE 6 Aldehydes (XIV)

a. Aldehyde XIVb (R²=n-Pr; R⁵=R⁶=Et): To a solution of alcohol XIIIb(2.34 g, 12.7 mmol) in wet CH₂Cl₂ (125 mL, prepared by stirring CH₂Cl₂with water and separating the organic layer) was added Dess-Martinperiodinane (8.06 g, 19.0 mmol). The mixture was stirred at roomtemperature for 40 min and was then poured into a mixture of 5% aqueousNa₂S₂O₃ (250 mL) containing 5.2 g NaHCO₃, and ether (200 mL). Thebiphasic mixture was stirred vigorously for 5 min and the aqueous layerwas extracted with 15% CH₂Cl₂/Et₂O (2×100 mL). The combined organiclayers were then washed with H₂O (3×75 mL) and brine, dried over MgSO₄,filtered and concentrated in vacuo to afford 2.06 g (88%) of desiredaldehyde XIVb, a clear colorless oil. ¹H NMR (300 MHz, CDCl₃) δ 9.60 (d,J=3.5 Hz, 1H), 3.49-3.30 (m, 5H), 1.96-1.85 (m, 2H), 1.39-1.31 (m, 2H),1.19 (t, J=7.1 Hz, 3H), 1.13 (t, J=7.1 Hz, 3H), 0.95 (t, J=7.3 Hz, 3H).

b. Aldehyde XIVb (R²=n-Pr; R⁵=R⁶=Et): To a solution of crude XIIIb (1.25g, 6.68 mmol) in a mixture of toluene (20 mL), ethyl acetate (20 mL),and water (3 mL) was added 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO),free radical (9 mg). The mixture was cooled to 0° C. and a sodiumhypochlorite solution, prepared by adding 4.3 mL of aqueous sodiumhypochlorite (10-13% available chlorine) to 1.6 g of NaHCO₃ in 20 mL ofwater, was added by portions over a period of 30 min. Sodium bromide(660 mg) was added and the solution turned pale orange. Within a fewminutes the color of the reaction mixture returned to off-white.Additional sodium hypochlorite (4.7 mL) was added in several portions todrive the reaction to completion. The aqueous layer was separated andextracted with toluene (20 mL) and ethyl acetate (2×20 mL). The combinedorganic extract was washed with a solution of KI (70 mg) in 10% aqueousKHSO₄. The organic layer was then washed with 5% Na₂S₂O₃ and pH 7phosphate buffer, dried (Na₂SO₄), and concentrated to give XIVb as apale yellow oil (1.1 g). Spectral data for this compound matched thatfor the product from Example 6a above.

c. Aldehyde XIVa (R²=Et; R⁵=Rf =Et): By a procedure analogous to thatdescribed for preparing alcohol XIVb, aldehyde XIVa was obtained in 80%yield. ¹H NMR (300 MHz, CDCl₃) δ 9.61 (d, J=3.6 Hz, 1H), 3.48-3.29 (m,5H), 2.02-1.90 (m, 2H), 1.19 (t, J=7.1 Hz, 3H), 1.14 (t, J=7.1 Hz, 3H),0.96 (t, J=7.4 Hz, 3H).

d. Aldehyde XIVc (R²=n-Bu; R⁵=R⁶=Et): By a procedure analogous to thatdescribed for preparing alcohol XIVb, aldehyde XIVc was obtained in 98%yield. H NMR (300 MHz, CDCl₃) δ 9.59 (d, J=3.6 Hz, 1H), 3.48-3.29 (m,5H), 1.97-1.87 (m, 2H), 1.39-1.22 (m, 4H), 1.18 (t, J=7.2 Hz, 3H), 1.13(t, J=7.2 Hz, 3H), 0.90 (t, J=7.0 Hz, 3H).

e. Aldehyde XIVd (R²=i-Bu; R¹=R⁶=Et): By a procedure analogous to thatdescribed for preparing alcohol XIVb, aldehyde XIVd was obtained in 96%yield. ¹H NMR (300 MHz, CDCl₃) δ 9.57 (d, J=3.7 Hz, 1H), 3.51-3.27 (m,5H), 1.83 (t, J=7.1 Hz, 3H), 1.66-1.55 (m, 1H), 1.20 (t, J=7.1 Hz, 3H),1.13 (t, J=7.1 Hz, 3H), 0.93 (d, J=6.6 Hz, 6H).

f. Aldehyde XIVe (R²=CH₂Ph; R¹=R⁶=Et): By a procedure analogous to thatdescribed for preparing alcohol XIVb, aldehyde XIVe was obtained in 97%yield. ¹H NMR (300 MHz, CDCl₃) δ 9.69 (d, J=2.9 Hz, 1H), 7.29-7.16 (m,5H), 3.65 (m, 1H), 3.53-3.42 (m, 1H), 3.30 (dd, J=13.5, 9.3 Hz, 1H),3.23-3.13 (m, 2H), 3.06-2.91 (m, 2H), 1.04 (t, J=7.1 Hz, 3H), 0.93 (t,J=7.1 Hz, 3H).

The preparation of clasto-lactacystin β-lactone and analogs thereofaccording to the synthetic scheme outlined in Scheme 1 as exemplified inExamples 7-9.

EXAMPLE 7 Aldol adducts (II)

a. Aldol adduct IIb (R²=n-Pr; R¹=i-Pr; R³=Me; R⁴=Ph; R⁵=R⁶=Et): To acold (−78° C.) solution of trans-oxazoline Ia (R¹=i-Pr; R⁴=Ph) in ether(35 mL) was added lithium bis(trimethylsilyl)amide (2.17 of a 1 Msolution in hexane, 2.17 mmol). After 30 min, the orange solution wastreated dropwise with a 1M solution of dimethylaluminum chloride inhexane (4.55 mL, 4.55 mmol) and the mixture was stirred for another 60min before being cooled down to −85° C. (liquid N₂ was added to the dryice/acetone bath). A solution of aldehyde XIVb (420 mg, 2.27 mmol) inether (4 mL) was then added over 10 min along the side of the flask. Themixture was then allowed to warm up to −40° C. over 2.5 h and thenquenched by adding 35 mL of saturated aqueous NH₄Cl and 25 mL AcOEt.Enough 2 N HCl was then added until 2 clear phases were obtained (ca. 15mL added). The aqueous layer was extracted with AcOEt (2×20 mL) and thecombined organic layers were washed successively with 0.5 N aqueous HCl(20 mL), H₂O (20 mL), 0.5 M aqueous NaHSO₃ (2×15 mL), saturated aqueousNaHCO₃ and finally with brine, then dried over Na₂SO₄ and concentratedin vacuo affording 879 mg (>100%) of crude Aldol product IIb which waspure enough to be used directly in the subsequent step. ¹H NMR (300 MHz,CDCl₃) δ 8.02-7.97 and 7.53-7.39 (m, 5H), 6.58 (d, J=9.9 Hz, 1H), 4.82(d, J=2.4 Hz, 1H), 3.73 (s, 3H), 3.69-3.61 (m, 2H), 3.49-3.39 (m, 2H),3.24-3.16 (m, 1H), 3.05 (m, 1H), 2.89 (m, 1H), 2.28-2.23 (m, 1H),1.98-1.91 (m, 1H), 1.37-1.20 (m, 6H), 1.19-1.06 (m, 6H), 0.87 (t, J=7.1Hz, 3H), 0.70 (d, J=6.7Hz, 3H).

Aldol product IIb was also obtained in 100% yield by a procedureanalogous to that described above but using cis-oxazoline Ib (see below)instead of trans-oxazoline Ia.

b. Aldol adduct IIb (R²=n-Pr; R¹=i-Pr; R³=Me; R⁴=Ph; R⁵=R⁶=Et): To acold (−78° C.) solution of trans-oxazoline Ia (R¹=i-Pr; R⁴=Ph) (20.74 g)in THF (280 mL) was added lithium bis(trimethylsilyl)amide (92.4 mL of a1 M solution in hexane) over 75 min. After 30 min, the orange solutionwas treated dropwise with a 1M solution of dimethylaluminum chloride inhexane (202 mL) and the mixture was stirred for another 40 min beforebeing cooled down to −85° C. (liquid N₂ was added to the dry ice/acetonebath). A solution of aldehyde XIVb (19.43 g) in THF (50 mL) was thenadded over 45 min. The mixture was then allowed to warm to −50° C. over40 min and then to −20° C. over 25 min. The yellow reaction mixture wasagain cooled to −78° C. and then quenched by cautious addition of40 mLof saturated aqueous NH₄Cl. The reaction mixture was poured slowly into460 mL of saturated aqueous NH₄Cl. AcOEt (500 mL) was added, and withgood stirring the reaction mixture was acidifed with 6 N HCl to producetwo clear phases. The aqueous layer was extracted with AcOEt (2×200 mL),and the combined organic layers were washed successively with H₂O (2×200mL), saturated aqueous NaHCO₃ (2×200 mL), and brine (2×300 mL). Theorganic extract was dried over Na₂SO₄ and MgSO₄ and concentrated invacuo to afford 41.55 g of crude Aldol product IIb which was pure enoughto be used directly in the subsequent step. Spectral data for thiscompound matched that for the product from Example 7a above.

c. Aldol adduct Ia (R²=Et; R=i-Pr; R³=Me; R⁴=Ph; R⁵=R⁶=Et): By aprocedure analogous to that described for preparing Aldol adduct IIb,the lithium anion of trans-oxazoline Ia (R¹=i-Pr; R⁴=Ph) was treatedsuccessively with dimethylaluminum chloride and aldehyde XIVa to provideAldol adduct IIa in 95% yield. ¹H NMR (300 MHz, CDCl₃) δ 8.00-7.97 and7.51-7.39 (m, 5H), 6.50 (d, J=9.9 Hz, 1H), 4.80 (d, J=2.4 Hz, 1H),3.81-3.64 (m, 2H), 3.74 (s, 3H), 3.45 (m, 2H), 3.19 (m, 2H), 2.93-2.84(m, 2H), 2.24 (m, 1H), 1.89 (m, 1H), 1.73-1.64 (m, 4H), 1.29 (t, J=7.2Hz, 3H), 1.12 (d, J=6.9 Hz, 3H), 1.07 (d, J=7.2 Hz, 3H), 0.70 (d, J=6.7Hz, 3H).

d. Aldol adduct IIc (R²=n-Bu; R¹=i-Pr; R³=Me; R⁴=Ph; R⁵=R⁶=Et). By aprocedure analogous to that described for preparing Aldol adduct IIb,the lithium anion of trans-oxazoline Ia (R¹=i-Pr; R⁴=Ph) was treatedsuccessively with dimethylaluminum chloride and aldehyde XIVc to provideAldol adduct IIc in 100% yield. ¹H NMR (300 MHz, CDCl₃) δ 8.02-7.98 and7.53-7.33 (m, 5H), 6.57 (d, J=10.0 Hz, 1H), 4.81 (d, J=2.3 Hz, 1H), 3.73(s, 3H), 3.68-3.60 (m, 2H), 3.49-3.17 (m, 2H), 3.00 (m, 1H), 2.90 (m,1H), 1.98-1.87 (m, 2H), 1.38-0.83 (m, 16H), 0.70 (d, J=6.7 Hz, 3H).

e. Aldol adduct IId (2=i-Bu; R¹=i-Pr; R³=Me; R⁴=Ph; R⁵=R⁶=Et): By aprocedure analogous to that described for preparing Aldol adduct IIb,the lithium anion of trans-oxazoline Ia (R¹=i-Pr; R⁴=Ph) was treatedsuccessively with dimethylaluminum chloride and aldehyde XIVd to provideAldol adduct IId in 100% yield. ¹H NMR (300 MHz, CDCl₃) δ 8.01-7.80 and7.55-7.20 (m, 5H), 4.87 (d, J=2.3 Hz, 1H), 3.73 (s, 3H), 3.69-3.58 (m,2H), 3.51-3.32 (m, 2H), 2.98-2.87 (m, 1H), 2.33-2.24 (m, 1H), 2.12-2.02(m, 1H), 1.83 (t, J=7.1 Hz, 1H), 1.35 (t, J=7.1 Hz, 3H), 1.25-1.05 (m,5H), 0.93 (d, J=6.6 Hz, 3H), 0.89 (d, J=6.5 Hz, 3H), 0.80 (d, J=6.5 Hz,3H), 0.69 (d, J=6.7 Hz, 3H).

f. Aldol adduct IIe (R²=CH₂Ph; R¹=i-Pr; R³=Me; R⁴=Ph; R⁵=R⁶=Et): By aprocedure analogous to that described for preparing Aldol adduct IIb,the lithium anion of trans-oxazoline Ia (R¹=i-Pr; R⁴=Ph) was treatedsuccessively with dimethylaluminum chloride and aldehyde XIVe to provideAldol adduct IIe in 100% yield. ¹H NMR (300 MHz, CDCl₃) δ 8.01-7.93 and7.54-7.10 (m, 10H), 4.71 (d, J=2.5 Hz, 1H), 3.73 (s, 3H), 3.68-3.58 (m,2H), 3.48-2.79 (m, 6H), 2.17 (m, 1H), 1.12-0.91 (m, 9H), 0.68 (d, J=6.7Hz, 3H).

EXAMPLE 8 γ-Lactams (IV)

a. γ-Lactam IVb (R²=n-Pr; R¹=i-Pr; R³=Me): A solution of Aldol adductIIb (4.72 g, 10.9 mmol) in 100mL 1:9 AcOH/MeOH, to which was added 4.8 g20% Pd(OH)₂/C, was vigorously shaken under 55 p.s.i. H₂ for 60 h. Themixture was brought down to atmospheric temperature before beingfiltered and concentrated in vacuo. The solid obtained was purified byflash chromatography (SiO₂, elution with 1% AcOH in 1:1 AcOEt/hexane)affording 2.23 g (75%) of desired γ-lactam IVb as a white solid. ¹H NMR(300 MHz, CDCl₃) δ 7.89 (br. s, 1H), 4.77 (br. d, J=11.5 Hz, 1H), 4.47(dd, J=11.5, 5.6 Hz, 1H), 4.08 (dd, J=9.4, 5.0 Hz, 1H), 3.83 (s, 3H),2.93 (m, 1H), 1.78-1.39 (m, 6H), 1.02-0.88 (m, 9H).

b. γ-Lactam IVa (R²=Et; R¹=i-Pr; R³=Me): By a procedure analogous tothat described for preparing γ-lactam IVb, Aldol adduct IIa washydrogenated at 55 p.s.i. for 48 h to provide γ-lactam IVa in 72% yield.¹H NMR (300 MHz, CDCl₃) δ 7.79 (br. s, 1H), 4.62 (br. d, J=11.2 Hz, 1H),4.51 (dd, J=11.2, 5.4 Hz, 1H), 3.83 (s, 3H), 2.85 (m, 11H), 1.77-1.64(m, 3H), 1.01 (t, J=7.4 Hz, 3H), 0.98 (d, J=6.9 Hz, 3H), 0.95 (d, J=6.9Hz, 3H).

c. γ-Lactam IVc (R²=n-Bu; R¹=i-Pr; R³=Me): A solution of Aldol adductIIc (361 mg, 0.80 mmol) in 6 mL 1:9 AcOH/MeOH, to which was added 250 mg20% Pd(OH)₂/C, was vigorously shaken under 50 p.s.i. H₂ for 24 h. Morecatalyst (100 mg) was then added and the mixture was again shaken at 50p.s.i. for another 24 h after which time it brought down to atmospherictemperature before being filtered. The filtrate was then heated toreflux for 30 min, cooled to room temperature and concentrated in vacuo.The solid obtained was co-evaporated once with toluene and purified byflash chromatography (SiO₂, elution with 4% MeOH/CHCl₃) affording 140 mg(61%) of desired γ-lactam IVc as a white solid. ¹H NMR (300 MHz, CDCl₃)δ 8.02 (br. s, 1H), 4.93 (br. d, J=11.3 Hz, 1H), 4.46 (dd, J=11.3, 5.5Hz, 1H), 4.15-4.08 (m, 1H), 3.83 (s, 3H), 2.94-2.87 (m, 1H), 1.80-1.34(m, 6H), 0.94 (d, J=6.9 Hz, 3H), 0.89 (t, J=7.2 Hz, 3H).

d. γ-Lactam IVd (R²=i-Bu; R¹=i-Pr; R³=Me): By a procedure analogous tothat described for preparing γ-lactam IVe, Aldol adduct IId washydrogenated at 50 p.s.i. for 40 h and heated to reflux for 30 minproviding γ-lactam IVd in 61% yield. ¹H NMR (300 MHz, CDCl₃) δ 7.92 (br.s, 1 H), 4.81 (br. d, J=11.5 Hz, 1H), 4.46 (m, 1H), 4.09 (m, 1H), 3.83(s, 3H), 3.04-2.98 (m, 1H), 1.78-1.73 (m, 2H), 1.66-1.47 (m, 3H),1.00-0.90 (m, 12H).

e. γ-Lactam IVe (R²=CH₂Ph; R¹=i-Pr; R³=Me): By a procedure analogous tothat described for preparing γ-lactam IVc, Aldol adduct IIe washydrogenated at 50 p.s.i. for 24 h and heated to reflux for 30 minproviding γ-lactam IVe in 71% yield. ¹H NMR (300 MHz, CDCl₃) δ 8.01 (br.s, 1H), 7.35-7.15 (m, 5H), 5.02 (br. d, J=11.7 Hz, 1H), 4.40-4.34 (m,1H), 4.06-4.01 (m, 1H), 3.84 (s, 3H), 3.34-3.27 (m, 1H), 3.10-3.04 (m,2H), 1.84-1.72 (m, 1H), 0.98 (d, J=6.7 Hz, 3H), 0.93 (d, J=6.9 Hz, 3H).

EXAMPLE 9 β-Lactones (VII)

a. β-Lactone VIIb (R²=n-Pr; R¹=i-Pr): To a cold (0° C.) solution ofγ-lactam IVb (2.20 g, 8.06 mmol) in EtOH (100 mL) was added 0.1N aqueousNaOH (100 mL, 10.0 mmol). The mixture was stirred at room temperaturefor 15 h after which time H₂O (50 mL) and AcOEt (100 mL) were added. Theaqueous layer was then washed with AcOEt (2×50 mL), acidified with 6Naqueous HCl and concentrated in vacuo to a volume of ca 60 mL. Thissolution was then frozen and lyophilized. The obtained solid wassuspended in THF, filtered to get rid of sodium chloride andconcentrated in vacuo affording 2.05 g (98%) of the desireddihydroxyacid as white solid. ¹H NMR (300 MHz, CD₃OD) δ 4.42 (d, J=5.8Hz, 1H), 3.90 (d, J=6.5 Hz, 1H), 2.84 (m, 1H), 1.70-1.24 (m, 6H),0.95-0.84 (m, 9H).

To a solution of the dihydroxyacid (1.90 g, 7.33 mmol) in anhydrous THF(36 mL) was added a solution of2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate(TBTU, 2.59, 8.06 mmol) in anhydrous MeCN (36 mL) followed bytriethylamine (0.72 mL, 22.0 mmol). After stirring for 70 min at roomtemperature, some toluene was added and the mixture was concentrated invacuo and co-evaporated 2 more times with toluene. Purification by flashchromatography (SiO₂, elution with 2:3 AcOEt/hexane) afforded 1.44 g(81%) of desired β-lactone VIb as a white solid. ¹H NMR (300 MHz, CDCl₃)δ 6.07 (br. s, 1H), 5.26 (d, J=6.1 Hz, 1H), 3.97 (dd, J=6.4, 4.4 Hz,1H), 2.70-2.63 (m, 1H), 2.03 (d, J=6.4 Hz, 3H), 1.93-1.44 (m, 5H), 1.07(d, J=7.0 Hz, 3H), 0.99 (d, J=7.3 Hz, 3H), 0.91 (d, J=6.7 Hz, 3H). LRMS(FAB) m/e 242 (M+H⁺).

b. β-Lactone VIIa (R²=Et; R¹=i-Pr): Hydrolysis of IVa, as described forIVb above, afforded the corresponding dihydroxyacid in 100% yield. ¹HNMR (300 MHz, CD₃OD) δ 4.45 (d, J=5.8 Hz, 1H), 3.90 (d, J=6.4 Hz, 1H),2.74 (m, 1H), 1.71-1.53 (m, 3H), 0.94 (t, J=7.4 Hz, 3H), 0.92 (d, J=6.8Hz, 3H), 0.88 (d, J=6.8 Hz, 3H).

By a procedure analogous to that described for preparing β-lactone VIIb,β-lactone VIIa was obtained in 79% yield. ¹H NMR (300 MHz, CDCl₃) δ 6.17(br. s, 1H), 5.30 (d, J=6.0 Hz, 1H), 3.98 (dd, J=6.4, 4.4 Hz, 1H), 2.60(m, 1H), 2.08 (d, J=6.4 Hz, 3H), 1.97 (m, 2H), 1.75 (m, 1H), 1.12 (t,J=7.5 Hz, 3H), 1.07 (d, J=6.8 Hz, 3H), 0.92 (d, J=6.8 Hz, 3H).

c. β-Lactone VIIc (R²=n-Bu; R¹=i-Pr): Hydrolysis of IVe, as describedfor IVb above, afforded the corresponding dihydroxyacid in 100% yield.¹H NMR (300 MHz, CD₃OD) δ 4.42 (d, J=5.8 Hz, 1H), 3.90 (d, J=6.4 Hz,1H), 2.86-2.79 (m, 1H), 1.70-1.24 (m, 8H), 0.97-0.86 (m, 9H).

By a procedure analogous to that described for preparing β-lactone VIIb,β-lactone VIIc was obtained in 40% yield. ¹H NMR (300 MHz, CDCl₃) δ 6.14(br. s, 1H), 5.27 (d, J=6.1 Hz, 1H), 3.97 (d, J=4.4 Hz, 1H), 2.68-2.61(m, 1H), 1.94-1.86 (m, 2H), 1.72-1.36 (m, 7H), 1.07 (d, J=7.0 Hz, 3H),0.93 (t, J=7.1 Hz, 3H), 0.91 (d, J=6.8 Hz, 3H). LRMS (FAB) m/e 256(M+H⁺)

d. β-Lactone VIId (R²=i-Bu; R¹=i-Pr): Hydrolysis of IVd, as describedfor IVb above, afforded the corresponding dihydroxyacid in 100% yield.¹H NMR (300 MHz, CD₃OD) δ 4.50 (d, J=5.8 Hz, 1H), 4.00 (d, J=6.5 Hz,1H), 3.09-3.02 (m, 1H), 1.90-1.61 (m, 3H), 1.49-1.40 (m, 2H), 1.02 (d,J=6.7 Hz, 3H), 0.98 (d, J=6.5 Hz, 3H), 0.97 (d, J=6.7 Hz, 3H).

By a procedure analogous to that described for preparing β-lactone VIIb,β-lactone VIId was obtained in 62% yield. ¹H NMR (300 MHz, CDCl₃) δ 6.16(br. s, 1H), 5.25 (d, J=6.1 Hz, 1H), 3.97 (d, J=4.4 Hz, 1H), 2.71 (dd,J=15.1, 6.2 Hz, 1H), 1.95-1.66 (m, 5H), 1.08 (d, J=6.9 Hz, 3H), 0.99 (d,J=6.3 Hz, 3H), 0.98 (d, J=6.3 Hz, 3H), 0.92 (d, J=6.7 Hz, 3H). LRMS(FAB) m/e 256 (M+H⁺).

e. β-Lactone VIIe (R²=CH₂Ph; R¹=i-Pr): Hydrolysis of IVe, as describedfor IVb above, afforded the corresponding dihydroxyacid in 88% yield. ¹HNMR (300 MHz, CD₃OD) δ 7.25-7.04 (m, 5H), 4.29 (d, J=5.7 Hz, 1H), 3.83(d, J=6.4 Hz, 1H), 3.01-2.82 (m, 3H), 1.65 (m, 1H), 0.90 (d, J=6.6 Hz,3H), 0.86 (d, J=6.8 Hz, 3H).

By a procedure analogous to that described for preparing β-lactone VIIb,β-lactone VIIe was obtained in 77% yield. ¹H NMR (300 MHz, CDCl₃) δ7.36-7.20 (m, 5H), 6.57 (br. s, 1H), 5.08 (d, J=5.4 Hz, 1H), 3.94 (d,J=4.5 Hz, 1H), 3.25 (d, J=10.1 Hz, 1H), 3.01-2.89 (m, 2H), 1.92-1.81 (m,1H), 1.05 (d, J=6.9 Hz, 3H), 0.86 (d, J=6.7 Hz, 3H). LRMS (FAB) m/e 290(M+H⁺).

The preparation of cis-oxazolines and trans-oxazolines according to thesynthetic schemes illustrated in Schemes 3 and 4 as illustrated byExamples 10 and 11.

EXAMPLE 10 cis-Oxazoline (Ia)

a. Ethyl 3-(isopropyl)propenoate (XV; R¹=i-Pr; R³=Me): To a stirredsolution of carbomethoxymethylene triphenylphosphorane (56.04 g, 167.6mmol) in dry CH₂Cl₂ (168 mL) at 0° C. was added dropwiseisobutyraldehyde (17.4 mL, 191.6 mmol). After 5 min, the reactionmixture was warmed to room temperature and stirred for 24 h. The solventwas removed in vacuo and pentane was added to the white oily solid toprecipitate triphenylphosphine oxide. The solid was filtered off and thefiltrate concentrated in vacuo. The procedure was repeated one more timeand the crude olefin (20.00 g, 93%) was obtained as a yellow oil thatwas sufficiently pure for the next step. ¹H NMR (300 MHz, CDCl₃) δ 6.95(dd, J=15.7, 6.6 Hz, 1H), 5.77 (dd, J=15.7, 1.5 Hz), 3.72 (s, 3H), 2.44(m, 1H), 1.06 (d, J=6.7 Hz, 6H).

b. (2S,3R)-Methyl 2,3-dihydroxy-3-[isopropyl]propionate (XVIa; R¹=i-Pr;R³=Me): A mixture of AD-mix-β (100.00 g,), methanesulfonamide (6.78 g,71.3 mmol) and tert-butanol-water (1:1, 720 mL) was stirred vigorouslyat room temperature for 5 min. The reaction mixture was then cooled to0° C. and α,β-unsaturated ester XV (R¹=i-Pr; R³=Me) (9.14 g, 71.3 mmol)was added dropwise via a Pasteur pipette. After stirring at 0° C. for 96h, Na₂SO₃ (3.0 g) was added, and stirring continued at room temperaturefor 1 h. The mixture was diluted with ethyl acetate (200 mL) andtransferred to a separatory funnel. The organic layer was removed andthe aqueous phase extracted with ethyl acetate (2×100 mL). The combinedorganic layers were dried (Na₂SO₄), filtered, and concentrated in vacuo.The yellow oil obtained was passed through a silica gel pad using 1:1hexane/ethyl acetate affording diol XVIa (R¹=i-Pr; R³=Me) (11.48 g, 94%)as a yellow solid. ¹H NMR (300 MHz, CDCl₃) δ 4.28 (dd, J=5.6, 1.8 Hz,1H), 3.80 (s, 3H), 3.48 (m, 1H), 3.28 (m, 1H), 2.33 (d, J=9.3 Hz, 1H),1.87 (m, 1H), 1.02 (d, J=6.7 Hz, 3H), 0.95 (d, J=6.7 Hz, 3H).

c. (2R,3R)-Methyl 2-bromo-3-dihydroxy-3-(isopropyl)propionate (XVIIa;R¹=i-Pr; R³=Me): (2S,3R)-Methyl 2,3-dihydroxy-3-[isopropyl]propionateXVIa (R¹=i-Pr; R³=Me) (1.0 g, 6.17 mmol) and trimethylorthobenzoate(1.02 mL, 80.1 mmol) were dissolved in CH₂Cl₂ (20 mL) and treated withBF₃-OEt₂ (40.0 μL, 0.32 mmol). After stirring for 75 min, the mixturewas concentrated under full vacuum (0.05 mm Hg) for 35 min. The mixturewas redissolved in CH₂Cl₂ (20.0 mL), cooled to 0° C. and treatedsequentially with Et₃N (43.0 μL, 0.31 mmol) and acetyl bromide (0.48 mL,6.49 mmol). After stirring for 4 h at 0° C., the reaction mixture wastreated with saturated NaHCO₃ solution (12 mL) and allowed to warm up toroom temperature. The layers were separated and the aqueous layer wasextracted with CH₂Cl₂ (2×20 mL). The combined organic layers were dried(Na₂SO₄), filtered and concentrated in vacuo affording the crude α-bromoβ-benzoate XVIIa (R¹=i-Pr; R³=Me) (1.36 g, 85%) as a clear colorlessoil. ¹H NMR (300 MHz, CDCl₃) δ 8.05-8.00 (m, 2H), 7.47-7.40 (m, 3H),5.57 (dd, J=8.8, 3.9 Hz, 1H), 4.47 (d, J=8.8 Hz, 1H), 3.67 (s, 3H), 2.45(m, 1H), 1.01 (d, J=6.8 Hz, 6H).

d. (2S3R)-Methyl 2-azo-3-dihydroxy-3-[isopropyl]propionate (XVIIIa;R¹=i-Pr; R³=Me): A solution of (2R, 3R)-Methyl2-bromo-3-dihydroxy-3-[isopropyl]propionate XVIIa (R¹=i-Pr; R³=Me) (2.00g, 6.07 mmol) in 15 mL DMSO was treated with sodium azide (790.0 mg,12.2 mmol). After stirring for 12 h at room temperature, the mixture waspartitioned between H₂O and ethyl acetate (50 mL each). The aqueouslayer was extracted with more ethyl acetate and the combined organiclayers were dried over MgSO₄ and concentrated in vacuo affording thedesired α-azo β-benzoate (1.55 g, 87%) as a yellow oil. ¹H NMR (300 MHz,CDCl₃) δ 8.07-8.02 (m, 2H), 7.55-7.43 (m, 3H), 5.40 (dd, J=8.8, 2.8 Hz,1H), 3.73 (s, 3H), 2.24 (m, 1H), 1.04 (d, J=5.8 Hz, 3H), 0.98 (d, J=5.8Hz, 3H).

Repeating the same procedure but using DMF as the solvent instead ofDMSO afforded the desired α-azo β-benzoate in 85% yield.

e. Benzamide XIXa (R¹=i-Pr; R³=Me): A solution of (2S,3R)-Methyl2-azo-3-dihydroxy-3-[isopropyl]propionate XVIIIa (R¹=i-Pr; R³=Me) (1.50g, 5.15 mmol) in ethyl acetate (25 mL) was treated with 200 mg of 20%Pd(OH)₂/C and the suspension was stirred vigorously in a H₂ atmosphereunder balloon pressure. After 12 hours, the mixture was filtered andrefluxed for 4 hours to complete the migration of the benzoyl group. Themixture was then cooled to room temperature and concentrated in vacuoaffording the desired benzamide (1.25 g, 92%) as a yellow oil. ¹H NMR(300 MHz, CDCl₃) δ 7.85-7.83 (m, 2H), 7.46-7.40 (m, 3H), 6.99 (br. d,J=9.1 Hz, 1H), 5.05 (dd, J=9.1, 1.9 Hz, 1H), 3.77 (s, 3H), 1.79 (m, 1H),1.03 (d, J=6.7 Hz, 3H), 0.99 (d, J=6.7 Hz, 3H).

f. cis-Oxazoline Ia (R¹=i-Pr; R³=Me): A solution of 500 mg of benzamideXIXa (R¹=i-Pr; R³=Me) (18.8 mmol) in CH₂Cl₂ (20 mL) was treated with4.50 mL thionyl chloride (61.7 mmol). After stirring at room temperaturefor 24 h, the mixture was diluted with CH₂Cl₂ and washed with saturatedNaHCO₃ solution, dried (Na₂SO₄), concentrated in vacuo andchromatographed (silica gel, 1:1 hexane/ethyl acetate) affording thedesired cis-oxazoline (248 mg, 53%) as a pale yellow oil. ¹H NMR (300MHz, CDCl₃) δ 8.01-7.97 (m, 2H), 7.52-7.38 (m, 3H), 4.94 (d, J=9.8 Hz,1H), 4.53 (dd, J=9.8, 7.8 Hz, 1H), 3.76 (s, 3H), 2.09 (m, 1H), 1.05 (d,J=6.5 Hz, 3H), 1.01 (d, J=6.7 Hz, 3H).

EXAMPLE 11 trans-Oxazoline (Ib)

a. Ethyl 3-(isopropyl)propenoate (XV; R¹=i-Pr; R³=Me): To a stirredsolution of carbomethoxymethylene triphenylphosphorane (56.04 g, 167.6mmol) in dry CH₂Cl₂ (168 mL) at 0° C. was added dropwiseisobutyraldehyde (17.4 mL, 191.6 mmol). After 5 min, the reactionmixture was warmed to room temperature and stirred for 24 h. The solventwas removed in vacuo and pentane was added to the white oily solid toprecipitate triphenylphosphine oxide. The solid was filtered off and thefiltrate concentrated in vacuo. The procedure was repeated one more timeand the crude olefin (20.00 g, 93%) was obtained as a yellow oil thatwas sufficiently pure for the next step. ¹H NMR (300 MHz, CDCl₃) δ 6.95(dd, J=15.7, 6.6 Hz, 1H), 5.77 (dd, J=15.7, 1.5 Hz), 3.72 (s, 3H), 2.44(m, 1H), 1.06 (d, J=6.7 Hz, 6H).

b. (2R, 3S)-Methyl 2,3-dihydroxy-3-[isopropyl]propionate (XVIb; R¹=i-Pr;R³=Me): To a clear yellow solution of K₂OsO₂(OH)₄ (246.1 mg, 0.67 mmol,0.95 mol %), hydroquinine 1,4-phthalazinediyl diether (555.1 mg, 0.71mmol, 1.01 mol %), N-methylmorpholine N-oxide (50 wt % in water, 25.0mL, 0.106 mol, 1.51 equiv.), t-BuOH (84 mL), and H₂O (58 mL) was addedat 25° C. the neat olefin XV (R¹=i-Pr; R³=Me) (9.0 g, 70.2 mmol) via asyringe pump over a period of 48 h (the syringe was connected to tubing,whose tip was immersed in the solution throughout the reaction time).The resulting clear orange solution was then stirred for another 60 min,after which time ethyl acetate (200 mL) and a solution of Na₂SO₃ (15.0g) in H₂O (150 mL) were added, and the resulting mixture was stirred for4 h. The phases were separated, and the aqueous layer was extracted withmore ethyl acetate (2×). The organic layers were then combined and thechiral ligand was extracted from the organic phase with a solution of0.3 M H₂SO₄ in saturated Na₂SO₄ (2×100 mL). The phases were once againseparated and the aqueous layer was extracted with more ethyl acetate(1×). The organic layers were combined and dried over Na₂SO₄, filteredand concentrated in vacuo. This afforded 11.4 g (ca. 100%) of a whiteoily solid which was shown to be 70% e.e (determined by ¹H NMR from a1:1 molar solution of diol and Europiumtris[3-(heptafluoropropylhydroxymethylene)-(−)-camphorate] in C₆D₆).Recrystallisation from 35-60° C. petroleum ether afforded 6.8 g (60%) of(2R,3S)-Methyl 2,3-dihydroxy-3-[isopropyl]propionate (XVIb; R¹=i-Pr;R³=Me) that was ca. 100% e.e., obtained as white crystals, mp=32-34° C.;=−110.6° (c 1.04, CHCl₃)]. ¹H NMR (300 MHz, CDCl₃) δ 4.28 (dd, J=5.6,1.8 Hz, 1H), 3.80 (s, 3H), 3.48 (m, 1H), 3.28 (m, 1H), 2.33 (d, J=9.3Hz, 1H), 1.87 (m, 1H), 1.02 (d, J=6.7 Hz, 3H), 0.95 (d, J=6.7 Hz, 3H).

c. (2S,3S)-Methyl 2-bromo-3-dihydroxy-3-(isopropyl)propionate (XVIIb;R¹=i-Pr; R³=Me): (2R,3S)-Methyl 2,3-dihydroxy-3-[isopropyl] propionateXVIb (R¹=i-Pr; R³=Me) (30.0 g, 185.2 mmol) and trimethylorthobenzoate(41.3 mL, 240.7 mmol) were dissolved in CH₂Cl₂ (400 mL) and treated withBF₃.OEt₂ (1.16 mL, 9.25 mmol). After 2 h, triethylamine (1.8 mL, 13mmol) was added, and the mixture was concentrated in vacuo and placedunder full vacuum (0.05 mm Hg) for 70 min. The residue was redissolvedin CH₂Cl₂ (400 mL), cooled to 0° C. and treated dropwise with acetylbromide (14.3 mL, 194.5 mmol). After 2 h, additional acetyl bromide(0.68 mL, 9.25 mmol) was added. After 30 min, saturated NaHCO₃ solution(500 mL) was added and the mixture was stirred vigorously for 5-10 min.The layers were separated and the aqueous layer was extracted withCH₂Cl₂ (2×20 mL). The combined organic layers were dried (Na₂SO₄),filtered and concentrated in vacuo affording the crude α-bromoβ-benzoate XVIIb (R¹=i-Pr; R³=Me) (66.23 g) as a clear colorless oil,containing ˜9.3% by wt. methyl benzoate. For product: ¹H NMR (300 MHz,CDCl₃) δ 8.05-8.00 (m, 2H), 7.47-7.40 (m, 3H), 5.57 (dd, J=8.8, 3.9 Hz,1H), 4.47 (d, J=8.8 Hz, 1H), 3.67 (s, 3H), 2.45 (m, 1H), 1.01 (d, J=6.8Hz, 6H).

d. (2R,3S)-Methyl2-azo-3-dihydroxy-3-[isopropyl]propionate (VIIIb;R¹=i-Pr; R³=Me): Sodium azide (24 g, 370 mmol) was added to 230 mL ofDMSO and the mixture was stirred at room temperature overnight. To theresultant solution was added a solution of (2S, 3S)-Methyl2-bromo-3-dihydroxy-3-[isopropyl] propionate (XVIIb; R¹=i-Pr; R³=Me) (61g, 185 mmol) in 20 mL DMSO. After stirring for 11 h at room temperature,the mixture was poured into water (1.5 L) and ether (200 mL) and stirredvigorously for 10-15 min. Ether (100 mL) was added and the layers wereseparated. The aqueous layer was extracted with ether (2×100 mL) and thecombined organic layers were washed with water (2×100 mL) and brine (100mL), dried over MgSO₄, and concentrated in vacuo affording the crudeproduct (57.5 g), containing approximately 3% starting material and 8%elimination byproduct. For product: ¹H NMR (300 MHz, CDCl₃) δ 8.07-8.02(m, 2H), 7.55-7.43 (m, 3H), 5.40 (dd, J=8.8, 2.8 Hz, 1H), 3.73 (s, 3H),2.24 (m, 1H), 1.04 (d, J=5.8 Hz, 3H), 0.98 (d, J=5.8 Hz, 3H).

e. Benzamide XIXb (R¹=i-Pr; R³=Me): To a cold (0-5° C.) solution of (2R,3S)-Methyl 2-azo-3-dihydroxy-3-[isopropyl]propionate XVIIIb (R¹=i-Pr;R³=Me) (55 g) in methanol (300 mL) was added 94 mL of 4 M HCl/dioxaneand 2.75 g of Pd(OH)₂/C. The mixture was purged with hydrogen andstirred at room temperature. The mixture was purged with hydrogen every30 min to remove the liberated nitrogen. After 4 h, the reaction mixturewas purged with nitrogen and additional Pd(OH)₂/C (1.3 g) was added. Thereaction mixture was purged with hydrogen and again purged every hourfor 4 h. The mixture was filtered and concentrated in vacuo. The residuewas dissolved in water and extracted with EtOAc. The aqueous layer wasbasified with Na₂CO₃ and again extracted with EtOAc. The combinedorganic extracts were washed with brine, dried over Na₂SO₄, andconcentrated to give a mixture of N- and O-benzoylated products, whichwas used directly in the next step.

f. trans-Oxazoline Ib (R¹=i-Pr; R³=Me): The crude product IXb obtainedin Example 10e above (37.3 g, 141 mmol) was dissolved in toluene (350mL). p-Toluenesulfonic acid (2.68 g, 14.1 mmol) was added and themixture was heated to reflux. Water was removed using a Dean Stark trap.After 3 h, 2.5 mL of water had been collected. The reaction mixture wascooled, diluted with EtOAc (100 mL), washed successively with saturatedNaHCO₃ (2×100 mL) and brine (100 mL), dried over MgSO₄, andconcentrated. The residue was purified over a pad of silica gel (˜400g), eluting with 25-30% EtOAc-hexanes to provide the trans-oxazoline 1b(R¹=i-Pr; R³=Me). ¹H NMR (300 MHz, CDCl₃) δ 8.01-7.97 (m, 2H), 7.52-7.38(m, 3H), 4.68 (apparent t, J=7 Hz, 1H), 4.57 (d, J=7 Hz, 1H), 3.81 (s,3H), 2.00-1.93 (m, 1H), 1.04 (d, J=6.7 Hz, 3H), 1.00 (d, J=6.8 Hz, 3H).

EXAMPLE 12 Inactivation of Proteasome Activity

Purification of 20S proteasome and proteasome activator PA28 wasperformed as previously described (Dick et al., J. Biol. Chem. 271:7273(1996)).

2 mL of assay buffer (20 mM HEPES, 0.5 mM EDTA, pH 8.0) andSuc-Leu-Leu-Val-Tyr-AMC in dimethyl sulfoxide were added to a 3 mLfluorescent cuvette, and the cuvette was placed in the jacketed cellholder of a Hitachi F-2000 fluorescence spectrophotometer. Thetemperature was maintained at 37° C. by a circulating water bath. 0.34mg of PA28 were added and the reaction progress was monitored by theincrease in fluorescence at 440 nm (λ_(ex)=380 nm) that accompaniesproduction of free AMC. The progress curves exhibited a lag phaselasting 1-2 min resulting from the slow formation of the 20S-PA28complex. After reaching a steady state of substrate hydrolysis,lactacystin was added to a final concentration of 1 mM, and the reactionwas monitored for 1 h. The fluorescence (F) versus time (t) data werecollected on a microcomputer using LAB CALC (Galactic) software.k_(inact) values were estimated by a nonlinear least-squares fit of thedata to the first order equation:

F=A(1−e ^(−kt))+C

where C=F=_(t=0) and A=F_(t=∞)−F_(t=0).

EXAMPLE 13 Inhibition of Intracellular Protein Degradation in C2C12Cells

C2C12 cells (a mouse myoblast line) were labeled for 48 hrs with³⁵S-methionine. The cells were then washed and preincubated for 2 hrs inthe same media supplemented with 2 mM unlabelled methionine. The mediawas removed and replaced with a fresh aliquot of the preincubation mediacontaining 50% serum, and a concentration of the compound to be tested.The media was then removed and made up to 10% TCA and centrifuged. TheTCA soluble radioactivity was counted. Inhibition of proteolysis wascalculated as the percent decrease in TCA soluble radioactivity. Fromthis data, an IC₅₀ for each compound was calculated.

EXAMPLE 14 Lactone Hydrolysis

The half-lives (t_(½)) for hydrolysis of β-lactone analogs to thecorresponding dihydroxy acids were measured at 37° C. at a concentrationof 200 mM in 20 mM HEPES, 0.5 mM EDTA, pH 7.8. Absorbance was measuredfor at least five half-lives (approximately 1 hour) at 230 nm, thewavelength at which there is the greatest difference in extinctioncoefficients for the lactone and dihydroxy. Half-lives were calculatedusing Guggenheim analysis (Gutfreund Enzymes: Physical Principles; Wileyand Sons: New York, 1975, pp 118-119). The results of Examples 12-14 arereported in Table 1.

TABLE 1 Kinetics of Inhibition of 20S Proteasome and Inhibition ofIntracellular Protein Degradation

Compound R² Kobs/[I] M⁻¹ s⁻¹)^(a) IC₅₀ (μM)^(b) t_(1/2) min^(c) 2 Me20,000 0.7-1.1 13 3a Et 39,000 0.32 15.3 3b n-Pr 46,500 0.29 15.3 3cn-Bu 38,000 0.33 17 3d i-Bu 17,000 0.51 16.8 3e CH₂Ph 6,400 — 6.8 3f OMe82,200 86 3.7 ^(a)Inactivation of the Chymotrypsin-like activity ofPA28-activated 20S proteasome. ^(b)Inhibition of intracellular proteindegradation in C2C12 cells. ^(c)Hydrolysis half-life

The results indicate that the compounds of the present invention arepotent inhibitors of the proteasome.

EXAMPLE 15 Reduction of Infarct Size and Neuronal Loss

Methods

Male Sprague Dawley rats (250-400 g) were anesthetized with haloethaneand subjected to middle cerebral artery (MCA) occlusion using a nylonfilament for 2 h. Subsequently, the filament was removed and reperfusionof the infarcted tissue occurred for 24 hours before the rat wassacrificed.

Immediately after the filament was withdrawn, the animals were evaluatedusing a neurological scoring system. Neurological scores were expressedon a scale from 0 to 10, with 0 representing no neurological deficit and10 representing severe neurological deficit. After 24 hours and beforesacrifice, animals were evaluated a second time using the sameneurological scoring system.

Staining of coronal sections (2.0 mm×7-8) with triphenyltetrazoliumchloride (TTC) taken throughout the brain were evaluated under blindedconditions using image analysis to determine infarct size.

Dosing Regimen

Rats were given i.v. bolus injections (1.0 mL/kg) of either vehicle (50%propylene glycol/saline; n=8) or 7-n-propyl-clasto-lactacystin β-lactone(3b) (0.3 mg/kg; n=7) at 2 hours after the start of the occlusion. Twoadditional groups of rats were given i.v. bolus injections (1.0 mL/kg)of 3b at 0 minutes, 2 hours, and 6 hours after the start of theocclusion. One group (0.1 mg/kg×3; n=6) received 0.1 mg/kg at each ofthese times, while another group (0.3 mg/kg×3; n=7) received 0.3 mg/kgat each of the three timepoints.

Results

In animals treated with a single dose of 7-n-propyl-clasto-lactacystinβ-lactone (3b), infarct volume was decreased by 50% (FIG. 1, 0.3×1).Infarct volume was not significantly decreased in either the 0.1 mg/kg×3dosage group or the 0.3 mg/kg×3 dosage group (FIG. 1).

All animals had a neurological score of 10±0 immediately after the 2hour ischemic episode. At 24 hours, the vehicle-treated rats had a meanscore of 8.7±0.6, whereas rats treated with a single 0.3 mg/kg dose of7-n-propyl-clasto-lactacystin β-lactone (3b) had a mean score of 4±1(FIG. 2). These data represent a 60% neurological improvement for thedrug-treated animals. No significant improvement in neurological scorewas observed in either the 0.1 mg/kg×3 dosage group of the 0.3 mg/g×3dosage group (FIG. 2).

Conclusion

7-n-propyl-clasto-lactacystin β-lactone, given once post-ischemia,provides significant protection in both the degree of neurologicaldeficit and infarcted brain damage. From these preliminary data, itappears that a single-dose regimen is preferred over a multiple-doseregimen.

Having now fully described this invention, it will be understood tothose of ordinary skill in the art that the same can be performed withina wide and equivalent range of conditions, formulations, and otherparameters without affecting the scope of the invention or anyembodiment thereof. All patents and publications cited herein are fullyincorporated by reference herein in their entirety.

What is claimed is:
 1. A process for forming anenantiomerically-enriched formyl amide of Formula XIV:

or a salt thereof, wherein R² is alkyl, cycloalkyl, aryl, alkaryl,aralkyl, alkoxy, hydroxy, alkoxyalkyl, or amido, where the ring portionof any of said aryl, aralkyl, or alkaryl can be optionally substituted;R⁵ and R⁶ are independently one of alkyl or alkaryl; or R⁵ and R⁶ whentaken together with the nitrogen atom to which they are attached form a5- to 7-membered heterocyclic ring, which can be optionally substituted,and which optionally include an additional oxygen or nitrogen atom; saidmethod comprising: (a) acylating an anion of a compound of Formula VIII:

where R⁸ is isopropyl or benzyl, with R²CH₂COCl to form anacyloxazolidinone of Formula IX:

where R² and R⁸ are as defined above; (b) stereoselectively reacting theacyloxazolidinone of Formula IX with benzyloxymethyl chloride to form aprotected alcohol of Formula X:

where R² and R⁸ are as defined above; (c) hydrolyzing the protectedalcohol of Formula X to form a carboxylic acid of Formula XI:

where R² is as defined above; (d) coupling said acid of Formula XI withan amine R⁵R⁶NH₂ to provide an amide of Formula XII:

where R², R⁵ and R⁶ are as defined above; (e) catalyticallyhydrogenating, the amide of Formula XII to form an alcohol of FormulaXIII:

where R², R⁵ and R⁶ are as defined above; and (f) oxidizing theresultant alcohol of Formula XIII to give a formyl amide of Formula XIV.2. The process of claim 1, wherein: R² is C₁₋₈alkyl, C₃₋₈cycloalkyl,C₂₋₈alkenyl, C₂₋₈alkynyl C₆₋₄aryl, C₆₋₁₀ ar(C₁₋₆)alkyl orC₁₋₆alk(C₆₋₁₀)aryl; and R⁵ and R⁶ are independently C₁₋₆alkyl,C₆₋₁₀ar(C₁₋₆)alkyl or C₁₋₆alk(C₆₋₁₀)aryl or together with the nitrogenatom to which they are attached form a 5- to 7-membered heterocyclewhich can be optionally substituted, and which optionally can include anadditional oxygen or nitrogen atom.